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Advances in Biotechnology
Indu Ravi Mamta BaunthiyalJyoti SaxenaEditors
Advances in Biotechnology
Indu Ravi • Mamta BaunthiyalJyoti SaxenaEditors
Advances inBiotechnology
123
EditorsIndu RaviIndira Gandhi National Open UniversityRegional Centre JaipurMansarovar, Jaipur, RajasthanIndia
Mamta BaunthiyalDepartment of BiotechnologyGovind Ballabh Pant EngineeringCollegeGhurdauri, Pauri, UttarakhandIndia
Jyoti SaxenaDepartment of Biochemical EngineeringBipin Tripathi Kumaon Institute ofTechnologyDwarahat, UttarakhandIndia
ISBN 978-81-322-1553-0 ISBN 978-81-322-1554-7 (eBook)DOI 10.1007/978-81-322-1554-7Springer New Delhi Heidelberg New York Dordrecht London
Library of Congress Control Number: 2013946949
� Springer India 2014This work is subject to copyright. All rights are reserved by the Publisher, whether the whole orpart of the material is concerned, specifically the rights of translation, reprinting, reuse ofillustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,and transmission or information storage and retrieval, electronic adaptation, computer software,or by similar or dissimilar methodology now known or hereafter developed. Exempted from thislegal reservation are brief excerpts in connection with reviews or scholarly analysis or materialsupplied specifically for the purpose of being entered and executed on a computer system, forexclusive use by the purchaser of the work. Duplication of this publication or parts thereof ispermitted only under the provisions of the Copyright Law of the Publisher’s location, in itscurrent version, and permission for use must always be obtained from Springer. Permissions foruse may be obtained through RightsLink at the Copyright Clearance Center. Violations areliable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, etc. in thispublication does not imply, even in the absence of a specific statement, that such names areexempt from the relevant protective laws and regulations and therefore free for general use.While the advice and information in this book are believed to be true and accurate at the date ofpublication, neither the authors nor the editors nor the publisher can accept any legalresponsibility for any errors or omissions that may be made. The publisher makes no warranty,express or implied, with respect to the material contained herein.
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Springer is part of Springer Science+Business Media (www.springer.com)
Foreword
The twenty-first century has been nicknamed as the era of biotechnol-
ogy. It has grown and evolved to such an extent over the past few years
that increasing numbers of professionals work in areas directly impacted
by it. It has been turned into a high science topic to our everyday
vocabulary over a short period of time.
It is quite remarkable to note how different branches of biotechnol-
ogy have emerged to have both substantial academic and industrial
impact in the not so distant future. The opportunities become wider and
the hopes brighter. Modern biotechnology has opened up many
opportunities in various sectors such as agriculture, food, forestry, waste
treatment, medicine, and pharmaceutical production. Covering even the
most important aspects of biotechnology in a single book that reaches
readers ranging from students to active researchers in academia and
industry is an enormous challenge. To prepare such a wide-ranging
book on biotechnology, editors have harnessed their own knowledge
and experience, gained in several departments and universities, and has
assembled experts to write chapters covering a wide array of biotech-
nology topics, including the latest advances. Advances in Biotechnology is
an important book that provides the information and insight to enable
readers to participate in the biotechnology debate. This book is intended
to serve both as a textbook for university courses as well as a reference
for researchers. It is increasingly important that scientists and engineers,
whatever their specialty, have a solid grounding in the fundamentals and
potential applications of biotechnology. The editors and their team are
to be warmly congratulated for bringing together this exclusive, timely,
and useful biotechnology book.
D. S. Chauhan Vice Chancellor
Uttarakhand Technical University, Dehradun
v
Preface
The twenty-first century looks to Biotechnology as the world’s fastest
growing and most rapidly changing technology that can improve the
human conditions. Modern biotechnology enables an organism to
produce a totally new product which the organism does not or cannot
produce in its normal course of life. The book Advances in Biotechnologyis a collection of topics on recent advances in certain ongoing biotech-
nological applications. Fourteen authoritative chapters on current
developments and future trends in biotechnology are empathized. The
book aims to cover a wide range of topics under all specialized domains
of microbial, plant, animal, and industrial biotechnology.
Chapter 1 provides a detailed account of high capacity vectors used
for various applications of genetic engineering. Chapter 2 is devoted to
the modern era DNA sequencing dealing with next generation
sequencing. Up-to-date methodological approaches such as use of
molecular markers (Chap. 3), DNA microarray technology (Chap. 6)
and proteomics (Chap. 8) have revolutionized biotechnology with a
wide array of applications in studies related to cancer biology, micro-
biology, plant science, environmental science, etc. Proteomics has
recently been of interest to scientists because it gives a better under-
standing of an organism than genomics.
Chapters 4, 5, 9, 11, and 12 are focused on the crucial role of bio-
technology in health care through gene therapy, gene silencing, stem cell
technology, monoclonal antibodies, and edible vaccines. Gene therapy is
being used for correcting defective genes that are responsible for disease
development; RNAi is a valuable research tool not only for functional
genomics, but also for gene-specific therapeutic activities. Monoclonal
antibodies are widely used for immunodiagnostic, immunotherapy, and
in biological and biochemical research. Key aspects of edible vaccines
like host plants, mechanism of action, advantages, limitations, and
different regulatory issues are contemplated upon.
In today’s world where products of microbial origin have proved
their utility in almost every sphere of life, metagenomic studies (Chap. 7)
have become highly important as they give a clue to the hidden wealth of
microbial world. Chapter 10 describes the utilization of biosensors in
various industries for monitoring food quality control, medical research,
clinical diagnosis, environmental monitoring, agriculture, bioprocesses,
vii
and control. Genes from microbes, plants, and animals are being used
successfully to enhance the ability of plants. Though improvement of
plants by genetic engineering opens up new possibilities to tolerate,
remove, and degrade pollutants, it is still in its research and develop-
ment phase with many technical issues needing to be addressed as
explained in Chap. 13. Finally, in Chap. 14, the great market potential
involved for biotechnological companies has been highlighted with
suggestions that can be set up for harnessing the vast potential involved
in biotech products all over the world.
This book is clearly a team effort, and many thanks are due. The
authors of the individual chapters have been chosen for their recognized
expertise and their contributions to the various fields of biotechnology.
Their willingness to impart their knowledge to their colleagues forms the
basis of this book and is gratefully acknowledged. Authors relied on
various sources, which are identified in the individual chapters. The
authors would also like to thank their colleague Ms. Shweta Ranghar
whose help during the preparation of this book was commendable.
Thanks are also due to Mr. Vikas Kumar for working on the illustra-
tions. The editors wish to thank their respective head of the institutions/
center for their encouragement and providing required ambience.
Moreover, this work would not have been brought to realization
without the prudence and the constant and conscientious support of the
publisher. We are grateful to Springer for publishing this book with
their customary excellence. Finally, special thanks go to our families,
who put up with longer hours, helpful suggestions, indispensable help,
and encouragement.
May 2013 Indu Ravi
Mamta Baunthiyal
Jyoti Saxena
viii Preface
Contents
1 High Capacity Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Bhakti Bajpai
2 DNA Sequencing: Method and Applications . . . . . . . . . . . 11Satpal Singh Bisht and Amrita Kumari Panda
3 Molecular Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Pavan Kumar Agrawal and Rahul Shrivastava
4 Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41K. Rohini
5 RNA Interference and Its Applications . . . . . . . . . . . . . . . 55Jyoti Saxena
6 DNA Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Ashwini M. Charpe
7 Metagenomics: The Exploration of UnculturableMicrobial World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105G. K. Joshi, J. Jugran and J. P. Bhatt
8 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117Indu Ravi
9 Recent Advances in Stem Cell Research . . . . . . . . . . . . . . 151Shweta Kulshreshtha and Pradeep Bhatnagar
10 Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179Mayank and Rachana Arya
11 Monoclonal Antibody Production and Applications . . . . . . 195B. D. Lakhchaura
12 Edible Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Jyoti Saxena and Shweta Rawat
ix
13 Engineering Plants for Phytoremediation . . . . . . . . . . . . . 227Mamta Baunthiyal
14 Future of Biotechnology Companies:A Global Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Sunita Chauhan and Pradeep Bhatnagar
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
x Contents
Contributors
Pavan Kumar Agrawal Govind Ballabh Pant Engineering College,
Ghurdauri, Pauri, Uttarakhand 246194, India , e-mail: p_k_agarwal@
rediffmail.com
Rachana Arya Electronics and Communication Engineering Depart-
ment, Bipin Tripathi Kumaon Institute of Technology, Dwarahat,
Uttarakhand 263653, India, e-mail: [email protected]
Bhakti Bajpai Biotechnology, ARIBAS, New Vallabh Vidya Nagar,
Gujarat 388121, India, e-mail: [email protected]
Mamta Baunthiyal Govind Ballabh Pant Engineering College, Ghurd-
auri, Pauri, Uttarakhand 246194, India , e-mail: mamtabaunthiyal@
yahoo.co.in
J. P. Bhatt Department of Zoology and Biotechnology, HNB Garhwal
University, Srinagar (Garhwal), Uttarakhand, India, e-mail: profjpbhatt
@gmail.com
Pradeep Bhatnagar Life Sciences, The IIS University, Mansarovar,
Jaipur, India, e-mail: [email protected]
Satpal Singh Bisht Department of Biotechnology, School of Life
Sciences, Mizoram University (A Central University), Tanhril, Aizawl,
Mizoram 796004, India, e-mail: [email protected]
Ashwini M. Charpe Plant Pathology Section, College of Agriculture,
Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra
444104, India, e-mail: [email protected]
Sunita Chauhan Kumarappa National Handmade Paper Institute,
Jaipur, Rajasthan, India, e-mail: [email protected]
G. K. Joshi Department of Zoology and Biotechnology, HNB Garhwal
University, Srinagar (Garhwal), Uttarakhand, India, e-mail: gkjoshi@
rediffmail.com
J. Jugran Department of Zoology and Biotechnology, HNB Garhwal
University, Srinagar (Garhwal), Uttarakhand, India, e-mail: jyotijugran
xi
Shweta Kulshreshtha Amity Institute of Biotechnology, Amity
University of Rajasthan, Jaipur, India, e-mail: shweta_kul17@
rediffmail.com
B. D. Lakhchaura Govind Ballabh Pant University of Agriculture and
Technology, Pantnagar, India, e-mail: [email protected]
Mayank Biochemical Engineering Department, Bipin Tripathi Kumaon
Institute of Technology, Dwarahat, Uttarakhand 263653, India, e-mail:
Amrita Kumari Panda Department of Biotechnology, Roland Institute
of Pharmaceutical Sciences, Berhampur, Orissa 760010, India , e-mail:
Indu Ravi IGNOU Regional Centre, Jaipur, Rajasthan, India, e-mail:
Shweta Rawat Biochemical Engineering Department, Bipin Tripathi
Kumaon Institute of Technology, Dwarahat, Uttarakhand 263653,
India, e-mail: [email protected]
K. Rohini Unit ofBiochemistry,Faculty ofMedicine,AIMSTUniversity,
3� Bukit Air Nasi, Jalan Semeling, 08100 Bedong, Kedah Darul Aman,
Malaysia, e-mail: [email protected]
Jyoti Saxena Biochemical Engineering Department, Bipin Tripathi
Kumaon Institute of Technology, Dwarahat, Uttarakhand 263653,
India, e-mail: [email protected]
Rahul Shrivastava Department of Biotechnology, Jaypee University of
Information Technology, Waknaghat, DumeharBani, Solan, Himachal
Pradesh, India, e-mail: [email protected]
xii Contributors
1High Capacity Vectors
Bhakti Bajpai
Abstract
Since the construction of the first generation of general cloning vectors inthe early 1970s, a large number of cloning vectors have been developed.Despite the bewildering choice of commercial and other availablevectors, the selection of cloning vector to be used can be decided byapplying a small number of criteria: insert size, copy number, incom-patibility, selectable marker cloning sites, and specialized vectorfunctions. Several of these criteria are dependent on each other. Mostgeneral cloning plasmids can carry a DNA insert up to around 15 kb insize. Several types of vectors are available for cloning large fragments ofDNA too. This chapter presents a consolidated account of some newgeneration of high-capacity vectors such as cosmid, yeast artificialchromosome (YAC), bacterial artificial chromosome (BAC), P1 phageartificial chromosome (PAC), and human artificial chromosome (HAC).
1.1 Introduction
A prime requisite for a gene cloning experimentis the selection of a suitable cloning vector, i.e.,a DNA molecule that acts as a vehicle for car-rying a foreign DNA fragment when insertedinto it and transports it into a host cell, which isusually a bacterium, though other types of livingcells can also be used. A wide variety of naturalreplicons exhibit the properties that allow themto act as cloning vectors, however, vectors may
also be designed to possess certain minimumqualification to function as an efficient agent fortransfer, maintenance, and amplification of tar-get DNA.
An ideal cloning vehicle would have thefollowing four properties:• Low-molecular weight• Ability to confer readily selectable phenotypic
traits on host cells• Single sites for a large number of restriction
endonucleases, preferably in genes with ascorable phenotype
• Ability to replicate within the host cell, so thatnumerous copies of the recombinant DNAmolecule can be produced and passed todaughter cells.B. Bajpai (&)
Biotechnology, ARIBAS, New Vallabh VidyaNagar, Gujarat, 388121, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_1, � Springer India 2014
1
In 1970s, when recombinant DNA technologywas being first developed, only a limited numberof vectors were available based on either high-copy number plasmids or phage k. Later phageM13 was developed as a specialist vector tofacilitate DNA sequencing; over a time a seriesof specialist vectors were constructed for specificpurpose. The examples of naturally occurring orartificially constructed vectors include vectorsbased on Escherichia coli plasmids, bacterio-phages (e.g., k, M13, P1), viruses (e.g., animalviruses—retrovirus, adenovirus, adeno-associ-ated virus, Herpes Simplex virus, Vaccinia virus,etc.; insect viruses—baculo virus; plant viru-ses—cauliflower mosaic virus, potato virus X,Gemini virus, etc.), Agrobacterium tumefaciensbased vectors, chimeric plasmids (e.g., cosmid,phagemid, phasmid, and fosmid), artificialchromosomes [e.g., YAC, BAC, PAC, MAC andHAC], and non-E. coli vectors (e.g., Bacillus andPseudomonas vectors etc.). Table 1.1 gives anidea about the size of the insert possible withdifferent types of vectors.
In order to determine the choice of vector fora particular cloning experiment, various factorsneed to be considered such as:1. Insert size: The insert size may vary for dif-
ferent types of vectors ranging from 5 to 25 kbfor plasmid vectors to[2,000 kb for HACs.
2. Vector size: The vector size range variesfrom 5 kb plasmid vectors to 6–10 megaba-ses HAC high-capacity vectors.
3. Restriction sites: The number of restrictionsites found in vectors is highly variable.There may be a few restriction sites in smallplasmid vectors but they may be increased bythe insertion of multiple cloning sites invectors.
4. Copy number: Different cloning vectors aremaintained at different copy numbers, depen-dent on the replicon of the plasmid. However, ahigh-copy number vector is desirable. Theorigin of replication determines the vectorcopy number, which could be in the range of25–50 copies/cell if the expression vector isderived from the low-copy number plasmidpBR322, or between 150 and 200 copies/cell,if derived from the high-copy number plasmidpUC.
5. Cloning efficiency: The ability to clone aDNA fragment inserted into a vector isknown as the cloning efficiency of the vector.
6. Ability to screen for inserts: For selection ofrecombinants, certain selectable markersshould be present in vectors in order to dis-tinguish them from non-recombinants.
7. Types of downstream experiments required.
1.2 Vectors for Cloning LargeFragments of DNA
1.2.1 Cosmid Vectors
Cosmids are hybrids between a phage DNAmolecule and a bacterial plasmid or are basicallya plasmid that carries a cos site, the substrate forenzymes that package k DNA molecule intophage coat proteins. The in vitro packagingreaction works not only with one genome butalso with any DNA molecule that carries cos siteseparated by 37–52 kb of DNA. It also needs aselectable marker, such as ampicillin resistancegene, and a plasmid origin of replication, ascosmids lack all the k genes, therefore do notproduce plaques. Instead colonies are formed onselective media, just as with a plasmid vector.The loading capacity of cosmids variesdepending on the size of the vector itself butusually lies around 40–45 kb—much more than
Table 1.1 Maximum DNA insert possible with differentcloning vectors
Vector Host Insertsize
Plasmid E.coli 5–25 kb
k phage E.coli 35–45 kb
P1 phage E.coli 70–100 kb
PACs E.coli 100–300kb
BACs E.coli \300 kb
YACs Saccharomycescerevisae
200–2000kb
HumanArtificialChromosomes(HACs)
CulturedHuman Cells
[2000kb
2 B. Bajpai
a phage k vector can accommodate. Afterpackaging in vitro, the particle is used to infectsuitable host. The recombinant cosmid DNA isinjected into the cell where it circularizes likephage DNA but replicates as a normal plasmidwithout the expression of any phage functions.Transformed cells are selected on the basis of avector drug resistance marker. The construct of atypical cosmid vector is shown in Fig. 1.1.
Cosmids provide an efficient means of clon-ing large pieces of DNA. Because of theircapacity to carry large fragments of DNA, cos-mids are particularly attractive for constructinglibraries of eukaryotic genome fragments. Partialdigestion with a restriction endonuclease pro-vided suitably large fragments. However, thereis potential problem associated with use of par-tial digests in this way. This is due to the pos-sibility of two or more genome fragmentsjoining together in the ligation reaction, hencecreating a clone containing fragments that werenot initially adjacent in the genome. The prob-lem can be overcome by the size fractionationand dephosphorylation of the foreign DNAfragments so as to prevent their ligation toge-ther. But this method is very sensitive to theexact ratio of target-to-vector DNAs becausevector-to-vector ligation can occur. Such diffi-culties have been overcome in a cosmid-cloningprocedure devised by Ish-Horowicz and Burke(1981). By appropriate treatment of the cosmidvector pJB8, left-hand and right-hand vectorends are purified which are incapable of self-
ligation but which accept dephosphorylatedforeign DNA. Thus, the method eliminates theneed to size the foreign DNA fragments andprevents formation of clones containing shortforeign DNA or multiple vector sequences.Figure 1.2 describes the cosmid-cloning proce-dure devised by Ish-Horowicz and Burke (1981).
Problems associated with lambda andcosmid cloning:1. Since repeats occur in eukaryotic DNA,
rearrangements can occur via recombinationof the repeats present on the DNA insertedinto lambda or cosmid.
2. Cosmids are difficult to maintain in a bacte-rial cell because they are somewhat unstable.
3. Not easy to handle due to its very large sizeof approximately 50 kb.
1.2.2 Yeast Artificial Chromosomes
A YAC is a vector used to clone DNA frag-ments larger than 100 kb and up to 3,000 kb.YACs are useful for the physical mapping ofcomplex genomes and for the cloning of largegenes. First described in 1983 by Murrayand Szostak, a YAC is an artificially con-structed chromosome that contains a centro-mere (CEN), telomeres (TEL), andan autonomous replicating sequence (ARS) ele-ment which are required for replication andpreservation of YAC in yeast cells. ARS ele-ments are thought to act as replication origins.A YAC is built using an initial circular plasmid,which is typically broken into two linear mole-cules using restriction enzymes. DNA ligase isthen used to ligate a sequence or gene of interestbetween the two linear molecules, forming asingle large linear piece of DNA.
A plasmid-derived origin of replication (ori)and an antibiotic resistance gene allow the YACvector to be amplified and selected forin E. coli. TRP1 and URA3 genes are includedin the YAC vector to provide a selection systemfor identifying transformed yeast cells thatinclude YAC by complementing recessive alle-les trp1 and ura3 in yeast host cell. YAC vectorcloning site for foreign DNA is located within
Fig. 1.1 Construct of a cosmid vector
1 High Capacity Vectors 3
the SUP4 gene. This gene compensates for amutation in the yeast host cell that causes theaccumulation of red pigment. The host cells arenormally red, and those transformed with YAConly, will form colorless colonies. Cloning of aforeign DNA fragment into the YAC causesinsertional inactivation, restoring the red color.Therefore, the colonies that contain the foreignDNA fragment are red.
1.2.2.1 Essential Components of YACVectors
1. Large DNA ([100 kb) is ligated between twoarms. Each arm ends with a yeast telomere sothat the product can be stabilized in the yeast
cell. Interestingly, larger YACs are morestable than shorter ones, which favors cloningof large stretches of DNA (Fig. 1.3a, b).
2. One arm contains an autonomous replicationsequence (ARS), a CEN, and TEL.
3. ampr for selective amplification and markerssuch as TRP1 and URA3 for identifying cellscontaining the YAC vector.
4. Recognition sites of restriction enzymes (e.g.,EcoRI and BamHI).
5. Insertion of DNA into the cloning site inac-tivates a mutant expressed in the vector DNAand red yeast colonies appear.
6. Transformants are identified as those redcolonies which grow in a yeast cell that ismutant for trp1 and ura3. This ensures thatthe cell has received an artificial chromosome
Fig. 1.2 Cosmid-cloningprocedure (Ish-Horowiczand Burke 1981)
4 B. Bajpai
with both TEL (because of complementationof the two mutants) and the artificial chro-mosome contains insert DNA (because thecell is red).The procedure for cloning in YAC is as given
below:1. The target DNA is partially digested by
EcoRI and the YAC vector is cleaved byEcoRI and BamHI.
2. The cleaved vector segment is ligated with adigested DNA fragment to form an artificialchromosome.
3. Yeast cells are transformed to make a largenumber of copies.
1.2.2.2 Advantages and DisadvantagesYeast expression vectors, such as YACs, yeastintegrating plasmids (YIps), and yeast episomalplasmids (YEps) have an advantage over bac-terial vectors (BACs) in that they can be used toexpress eukaryotic proteins that require post-translational modification. However, YACs aresignificantly less stable than BACs, producing‘‘chimeric effects’’: artifacts where the sequenceof the cloned DNA actually corresponds not to asingle genomic region but to multiple regions.Chimerism may be due to either coligation ofmultiple genomic segments into a single YAC,
or recombination of two or more YACs trans-formed in the same host yeast cell. The inci-dence of chimerism may be as high as50 %. Other artifacts are deletion of segmentsfrom a cloned region, and rearrangement ofgenomic segments (such as inversion). In allthese cases, the sequence as determined from theYAC clone is different from the original, naturalsequence, leading to inconsistent results, anderrors in interpretation if the clone’s informationis relied upon. Due to these issues, the HumanGenome Project ultimately abandoned the use ofYACs and switched to BACs, where the inci-dence of these artifacts is very low.
1.2.3 Bacterial Artificial Chromosome
As the Human Genome Project was underway inthe early 1990s, there was a need to create high-resolution physical map of each human chro-mosome, which would permit the isolation ofshort DNA fragments for direct sequencing andother manipulations. In response to this, theYAC system was developed. Although yeast cancarry the DNA as large as one Mb, subsequentstudies indicated that yeast system presentedseveral difficulties in the creation of a humangenome map. Additionally, yeast cells were not
Fig. 1.3 a Linear form ofyeast artificialchromosome. b Circularform of yeast artificialchromosome
1 High Capacity Vectors 5
as familiar to molecular biologist as E. coli. Tocircumvent these difficulties, a bacterial cloningsystem based on the well-characterized E. coli Ffactor, a low-copy plasmid that exist in asupercoiled form was developed by HiroakiShizuya in 1992.
A BAC is a DNA construct, based on afunctional fertility plasmid (or F-plasmid), usedfor transforming and cloning in bacteria, usu-ally E. coli. F-plasmids play a crucial rolebecause they contain partition genes that pro-mote the even distribution of plasmids afterbacterial cell division. The BAC’s usual insertsize is 150–350 kb. The replication of F factor isstrictly controlled by the regulatory functions ofE. coli; as a result F factor is maintained as alow-copy number (i.e., one or two copies percell). This allows stable maintenance of largeDNA inserts and reduces the potential forrecombination between DNA fragments carriedby the vector, which was a limitation observedwith cosmid-cloning system. In addition to sta-ble maintenance, the structural stability of F-factors allows complex genomic DNA inserts tobe maintained with a great degree of structuralstability in the E. coli host. The structure of atypical BAC is given in Fig. 1.4.
BACs have several advantages over YACs. Itwas observed that a large percentage of YACscarried chimeric inserts, making mapping efforts
confusing and difficult. BACs in contrast arevirtually free from chimerism. Another problemwith YAC is that multiple YAC chromosomesmay coexist in a single yeast cell, whereas in theBAC system the F factor encoded parA and parBgene are involved in exclusion of multipleF-factors, as a result multiple F-factors cannotcoexist in a single cell.
1.2.3.1 BAC Vector Cloning SiteThe cloning segment of BAC vector includes(1) two bacteriophage markers lambda cosN andP1 loxP, (2) three restriction enzyme sites(EcoRI, HindIII, and BamHI) for cloning, and(3) a GC- rich NotI restriction enzyme site forpotential excision of inserts. The cosN site pro-vides a fixed position for cleavage by bacterio-phage lambda enzyme terminase, which allowsthe convenient generation of a linear form of theBAC DNA. The cosN site is also used to pack-age approximately 50 kb DNA into bacterio-phage lambda head as a particle. The methodknown as Fosmid for F-based cosmid system isextremely efficient, thus very useful when DNAis precious or available in small amounts. TheP1 loxP site allows the retrofitting of additionalcomponents to BAC vector at a later stage. TheloxP site is also utilized to linearize BACsthrough the P1 phage protein Cre, which catal-yses strand exchange between two DNA strandsat the loxP sites.
1.2.3.2 Uses
Inherited Disease
BACs are now being utilized to a greater extentin modeling genetic diseases, often along-side transgenic mice. BACs have been useful inthis field as complex genes may have severalregulatory sequences upstream of the encodingsequence, including various promoter sequencesthat will govern a gene’s expression level. BACshave been used to some degree of success withmice while studying neurological diseases suchas Alzheimer’s disease or as in the caseof aneuploidy associated with Down syndrome.Fig. 1.4 Bacterial artificial chromosome
6 B. Bajpai
There have also been instances when they havebeen used to study specific oncogenes associ-ated with cancers. They are transferred over tothese genetic disease models by electroporation/transformation, transfection with a suitable virusor microinjection. BACs can also be utilized todetect genes or large sequences of interest andthen used to map them onto the human chro-mosome using BAC arrays. BACs are preferredfor these kinds of genetic studies because theyaccommodate much larger sequences withoutthe risk of rearrangement, therefore more stablethan other types of cloning vectors.
Infectious Diseases
The genomes of several large DNA and RNAviruses have been cloned as BACs. These con-structs are referred to as ‘‘infectious clones,’’ astransfection of the BAC construct into host cells issufficient to initiate viral infection. The infectiousproperty of these BACs has made the study ofmany viruses such as the herpes viruses, poxvi-ruses, and coronaviruses more accessible.Molecular studies of these viruses can now beachieved using genetic approaches to mutate theBAC while it resides in bacteria. Such geneticapproaches rely on either linear or circular tar-geting vectors to carry out homologousrecombination.
Genome Sequencing
BACs are often used to sequence the genome oforganisms in genome projects, for examplethe Human Genome Project. A short piece of theorganism’s DNA is amplified as an insert inBACs, and then sequenced. Finally, thesequenced parts are rearranged in silico, result-ing in the genomic sequence of the organism.
1.2.4 P1 Phage Derived ArtificialChromosome
The P1-derived artificial chromosomes are DNAconstructs derived from the DNA of P1 bacte-riophage and BAC. They can carry large amounts(about 100–300 kb) of other sequences for avariety of bioengineering purposes. It is one type
of vector used to clone DNA fragments (100- to300-kb insert size; average, 150 kb)in E. coli cells. PACs have a low-copy numberorigin of replication based on P1 bacteriophage,which is used for propagation. Similar to BACs,PACs allow replication of the clones at one copyper cell and replicate clones across 60–100 gen-erations. In contrast to BACs, PACs have a neg-ative selection against non-recombinants. PACsalso have an IPTG- inducible high-copy numberorigin of replication that can be utilized for DNAproduction. These can accommodate largerinserts of DNA than a plasmid or many other typesof vectors. Sometimes, the number of inserts canbe as high as 300 kb (Fig. 1.5).
1.2.4.1 Uniqueness of P1-bacteriophage
A P1 phage can exist in both lysogenic and lyticforms in the host cell, but its unique feature liesin its existence as an independent entity withinthe cell, rather than incorporating itself with thehost chromosomes during the phase of ‘lysog-eny’. Thus, it acts like a plasmid during itsexistence and can replace the function of aplasmid during processes, which entails thisfeature. However, the scientists consider P1derived chromosome to contain features of bothplasmids and ‘F’ factor, which is a unique plas-mid like DNA sequence used in creating BAC.
In comparison with YACs, PACs offer certainadvantages: (1) these are bacterial systems thatare easy to manipulate, (2) libraries are gener-ated using bacterial hosts with well definedproperties, (3) transformation efficiency ishigher than that obtained by YACs, (4) PACs arenonchimeric, and (5) PACs have very stableinserts and do not delete sequences.
1.2.4.2 Construction of PACs ThroughElectroporation
During the construction of PACs, P1 phagecontaining cells will undergo a process known as‘electroporation’, which will increase the per-meability of the cell membrane and allow DNAmaterial to enter the cell and couple with the
1 High Capacity Vectors 7
existing DNA. This process will give rise toPACs and from there onwards, the PACs canreplicate within the cell through ‘lysogeny’,without destructing the cell or incorporating intorest of the chromosomes.
1.2.4.3 Uses of PACsPACs are in high demand for cloning importantbiomedical sequences, which are essential formany scientific functions. One of its main uses isthe genome analysis and map-based cloning ofcomplex plants and animals, which requiresisolation of large pieces of DNA rather thansmaller segments. Furthermore, PAC-basedcloning is useful in the study of ‘phage therapy’and in scientific studies focusing on how anti-biotics act on a particular bacteria.
Although there are other forms of artificialchromosomes which can accommodate morebase pairs than PACs, relative user friendlinessof these vectors makes them a popular choiceamong many biomedical researchers.
1.2.5 Human Artificial Chromosomes
The idea of using artificial chromosomes aspotential vectors for gene therapy applicationscame as a consequence of the first studies
involving chromosome manipulation, designedto understand human chromosome structure, andto identify elements necessary for their correctfunctioning. There are two approaches that canbe used for the development of artificial chro-mosomes: top-down approach in which naturalchromosomes are truncated by radiation ortelomere-associated fragmentation; and the bot-tom-up approach in which a de novo chromo-some is formed from the basic elements ofCENs, TEL, and origins of replication.
The construction of YAC showed CENs,TEL, and origins of replication as the elementsnecessary for extrachromosomal retention andled to the development of mammalian chromo-somes which are similar to yeast chromo-somes. Many experiments designed to findmammalian origins of replication could notidentify specific sequences responsible formammalian genome replication. However, thestructure of human TEL was soon described as anarray. The confirmation came from the observa-tion of newly formed TEL in the a globin gene,caused by insertion of (TTAGGG)n sequence.The discovery of the telomeric sequence as atandemly repeated (TTAGGG)n sequence orien-tated 50-30 toward the end of the chromosome andits role in telomere formation led to the devel-opment of telomere-mediated chromosomefragmentation (top-down approach), which
Fig. 1.5 Phage artificial chromosome
8 B. Bajpai
allowed the isolation of minichromosomes insomatic cell hybrid. The first ‘‘top down’’approach or TACF (telomere-associated chro-mosome fragmentation) involved modifyingnatural chromosome into smaller defined mini-chromosomes in cultured cells. Followingrecombination and subsequent breakage betweenhomologous sequences on the endogenous hostchromosome and an incoming telomere con-taining the targeting vector, engineered mini-chromosomes as small as 450 kb in size in aviancells have been generated. The approach hasbeen important for studying the structure,sequence organization, and size requirements ofthe human X and Y chromosomes.
Second approach, the ‘‘bottom up’’ orassembly approach involved generating HACs inhuman cells by introducing defined chromo-somal sequences as naked DNA includinghuman TEL, alpha satellite (alphoid) DNA andgenomic fragmentation containing replicationorigins. The de novo HACs are generated fol-lowing recombination and some amplification ofthe input DNA within the host cell. Together, thegeneration of minichromosomes and de novoHACs has identified alphoid DNA as the majorsequence element of the CEN and determinedthe minimum size (*700–100 kb) required forCEN function and stability.
Second approach includes the generation ofSATACs (satellite DNA-based artificial chro-mosomes) following integration of repetitiveDNA into preexisting centromeric regions ofhost chromosomes and modifying small humanmarker chromosomes (minichromosomesderived from naturally occurring chromosomes).The two approaches are shown in Fig. 1.6.
The de novo HACs when introduced into thecell, undergo a process of recombination andamplification forming large (1–10 Mb) circularmolecules (usually at one or two copies per cell)which are mitotically stable in the absence ofany selection for 9 months in some cells. Theefficiency of de novo HAC formation and sta-bility depends on the presence of a CEN proteinB-binding sequence (CENP-B box) and, to someextent, on the chromosomal origin of the alphoidtemplate and the longer length of the alphoidarray ([100 bp).
Established HACs can be either in a linear ora circular state. PAC-based constructs carrying*70 kb of alphoid DNA array with or withouttelomeric sequences and in circular or linearizedstate were used to transfect by lipofectionHT1080 cells. Circular alphoid DNA vectorsestablished effectively as minichromosomes inany condition, demonstrating that TEL are notrequired for the circular conformation. However,
Fig. 1.6 Structural map of‘top-down’ engineeredmammalian artificialchromosome systems
1 High Capacity Vectors 9
capped TEL were essential for establishment oflinear PAC vectors because these vectorsshowed poor chromosome formation in theirabsence.
1.2.5.1 Advantages and Uses of HACHuman artificial chromosomes (HACs) repre-sent another extrachromosomal gene deliveryand gene expression vector system. Althoughthis technology is less advanced than virusderived vectors, HACs have several potentialadvantages over currently used episomal viralvectors for gene therapy applications. Thepresence of a functional CEN provides a long-term stable maintenance of a HAC as a singlecopy episome without integration to the hostchromosomes. There is no upper size limit toDNA that should be cloned in HAC that allowsthe use of complete genomic loci, including theupstream and downstream regulatory elements.Additionally, being solely human in origin,HAC vectors cannot evoke adverse host immu-nogenic responses or induce any risk of cellulartransformation.
HAC-based vectors offer a promising systemfor delivery and expression of full-length humangenes of any size.
1.3 Conclusion
A vector is a DNA molecule used as a vehicle totransfer foreign genetic material into anothercell. All engineered vectors have an origin of
replication, a multicloning site, and a selectablemarker. Genome size varies among differentorganisms and the cloning vector must beselected accordingly. For a large genome, avector with a large capacity is chosen so that arelatively small number of clones are sufficientfor coverage of the entire genome. However, it isoften more difficult to characterize an insertcontained in a high capacity vector. The devel-opment of extrachromosomal large-capacitycloning vectors for mammalian cells represents apowerful tool for functional genomic studies.Further, the advances in genome library con-struction and DNA sequencing are mainly due tothe development of high capacity vectors such ascosmids, BACs, PACs, YACs, and HACs.
References
Ish-Horowicz D, Burke JF (1981) Rapid and efficientcosmid cloning. Nucleic Acids Res 9(13):2989–2998
Murray AW, Szostak JW (1983) Construction of artificialchromosomes in yeast. Nature 305(5931):189–193
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T,Tachiiri Y, Simon M (1992) Cloning and stablemaintenance of 300-kilobase-pair fragments ofhuman DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci 89:8794–8797
10 B. Bajpai
2DNA Sequencing: Methodsand Applications
Satpal Singh Bisht and Amrita Kumari Panda
Abstract
Determination of the precise order of nucleotides within a DNA moleculeis popularly known as DNA sequencing. About three decades ago in theyear 1977, Sanger and Maxam–Gilbert made a breakthrough thatrevolutionized the world of biological sciences by sequencing the5,386-base bacteriophage uX174. From the year 1977 to till date DNAsequencing came across much advancement in terms of sequencing toolsand techniques. The modern era DNA sequencing are dealing with Nextgeneration sequencing and many other advancement are available to theresearchers, practitioners, and academicians at a very reasonable costwith highest accuracy. The biological databases are being flooded with ahuge flow of sequences coming out from various organisms across theworld. Today the researchers and scientists across the various fields areutilizing these data for a variety of applications including food securityby developing better crops and crop yields, livestock, improveddiagnostics, prognostics, and therapies for many complex diseases.
2.1 Introduction
DNA is the blueprint of life consisting ofchemical building blocks called nucleotides.These building blocks are made of three parts:
phosphate, sugar group, and one of the four typesof nitrogen bases viz Adenine (A), Thymine (T),Guanine (G), or Cytosine (C). To form a strandof DNA, nucleotides are linked into chains, withthe phosphate and sugar groups alternately. Theorder or sequence of these bases determines whatbiological instructions are contained in a strandof DNA. For example, the sequence ATCGTTmight instruct for blue eyes, while ATCGCT forbrown. Each DNA sequence that containsinstructions to make a protein is known as gene.The size of a gene may vary greatly, rangingfrom about 1,000 bases to 2300 kilo bases inhumans. DNA has double helical structure inwhich two strands run in opposite directions.Each ‘‘rung’’ of the ladder is made up of two
S. S. Bisht (&)Biotechnology School of Life Sciences, MizoramUniversity, Tanhril, Mizoram, Aizawl, 796004,Indiae-mail: [email protected]
A. K. PandaBiotechnology, Roland Institute of PharmaceuticalSciences, Orissa, Berhampur, 760010, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_2, � Springer India 2014
11
nitrogen bases; paired together by hydrogenbonds, because of the highly specific nature ofthis type of chemical pairing, base A always pairswith base T, and likewise C with G. Therefore, ifthe sequence of the bases on one strand of a DNAdouble helix is known, it is simple to figure outthe sequence of bases on the other strand.
The most significant advances in genetics dur-ing 1990s have come from complete sequencing ofchromosomes. The first eukaryotic chromosome tobe completely sequenced was chromosome III ofSaccharomyces cerevisiae, published in 1992. Thiswas followed by the first complete genomesequence for a free living organism, the bacteriumHaemophilus influenzae in the year 1995 and thefirst complete sequence of an eukaryotic genome S.cerevisiae in 1996. Later the complete genomicsequences of important model organisms such asEscherichia coli, the nematode Coenarhabditiselegans, the fruit fly Drosophila, and the plantArabidopsis became available. Genome projectsfor many organisms have either been completed orwill be completed shortly, such as Palaeo-Eskimo,an ancient-human, Neanderthal Homo neander-thalensis (partial), Neanderthal genome project,Common Chimpanzee Pan troglodytes; Chim-panzee genome project, Domestic cow, Bovinegenome, Honey-bee genome sequencing consor-tium, Human microbiome project, Internationalgrape genome program, International HapMapproject including Human genome project whichhas now entered into functional genomics phase.The main objective of most genome projects is todetermine the DNA sequence of the entire genomeor of its large number of transcripts. This leads tothe identification of all or most of the genes and tocharacterize various structural features of thegenome. In many molecular biology laboratoriesDNA sequencing is chiefly used to characterizenewly cloned cDNAs to confirm the identity of aclone or mutation, to check the fidelity of a newlycreated mutation, PCR products and screening toolto identify polymorphism. Now-a-days, by theadvent of automated DNA sequencing and Nextgeneration sequencing (NGS) complete genomesequencing data of many organisms are availablefor genetic studies.
2.2 Landmarks in DNA Sequencing
• 1953 Discovery of the structure of the DNAdouble helix.
• 1972 Development of recombinant DNAtechnology.
• 1977 The first complete genome of bacterio-phage uX174 sequenced.
• 1977 Allan Maxam and Walter Gilbert publish‘‘DNA sequencing by chemical degradation.’’
• 1984 Medical Research Council scientistsdecipher the complete DNA sequence of theEpstein-Barr virus, 170 kb.
• 1986 Leroy E. Hood’s laboratory at the Cali-fornia Institute of Technology and Smithannounced the first semi-automated DNAsequencing machine.
• 1987 Applied Biosystems marketed first auto-mated sequencing machine, the model ABI 370.
• 1990 The U.S. National Institutes of Health(NIH) begins large-scale sequencing trials onMycoplasma capricolum, E. coli, C. elegans,and S. cerevisiae.
• 1991 Sequencing of human expressedsequence tags begins in Craig Venter’s lab.
• 1995 Craig Venter, Hamilton Smith, andcolleagues at The Institute for GenomicResearch (TIGR) published the first completegenome of a free-living organism, the bacte-rium Haemophilus influenzae.
• 1996 Pal Nyren and his student Mostafa Ro-naghi at the Royal Institute of Technology inStockholm published their method ofpyrosequencing.
• 1998 Phil Green and Brent Ewing of theUniversity of Washington publish ‘‘phred’’ forsequencer data analysis.
• 2000 Lynx Therapeutics publishes and markets‘‘MPSS’’—a parallelized, adapter/ligation-mediated, bead-based sequencing technology,launching ‘‘next-generation’’ sequencing.
• 2001 A draft sequence of the human genomepublished.
• 2004 454 Life Sciences markets a parallelizedversion of pyrosequencing. The first version oftheir machine reduced sequencing costs 6-foldcompared to automated Sanger sequencing,
12 S. S. Bisht and A. K. Panda
and was the second of a new generation ofsequencing technologies, after MPSS.
• 2005 Solexa/ Illumina sequence analyzerwhich gave an output data of 10E+7 Kbp.
• 2010 Illumina Hi-seq 2000 was introducedwhich gave an output of 10E+8 Kbp.
2.3 Sequencing Methods
2.3.1 Maxam–Gilbert Method
Allan Maxam and Walter Gilbert developed amethod for sequencing single-stranded DNA bya two-step catalytic process involving piperidineand two chemicals that selectively attack purinesand pyrimidines (Maxam and Gilbert 1977).Purines react with dimethyl sulfate and pyrimi-dines react with hydrazine in such a way so as tobreak the glycoside bond between the ribosesugar and the base, displacing the base. Piperi-dine catalyzes cleavage of phosphodiester bondswhere the base has been displaced. Moreover,dimethyl sulfate and piperidine alone selectivelycleave guanine nucleotides but dimethyl sulfateand piperidine in formic acid cleave bothguanine and adenine nucleotides. Similarly,hydrazine and piperidine cleave both thymineand cytosine nucleotides, whereas hydrazine andpiperidine in 1.5 M NaCl only cleave cytosine
nucleotides. The use of these selective reactionsto DNA sequencing involves creating a single-stranded DNA substrate carrying a radioactivelabel on the 50 end. This labeled substrate issubjected to four separate cleavage reactions,each of which creates a population of labeledcleavage products ending in known nucleotides.The reactions are loaded on high percentagepolyacrylamide gels and the fragments areresolved by electrophoresis. The gel then istransferred to a light-proof X-ray film cassette, apiece of X-ray film placed over the gel, and thecassette placed in a freezer for several days.Wherever a labeled fragment stopped on the gel,the radioactive tag would expose the film due toparticle decay (autoradiography).
The dark autoradiography bands on the filmrepresent the 50 to 30 DNA sequence when readfrom bottom to top (Fig. 2.1). The process ofbase calling involves interpreting the bandingpattern relative to the four chemical reactions.For example, a band in the lanes correspondingto the C only and the C ? T reactions called a C.If the band present in the C ? T reaction lane butnot in the C reaction lane it is called as T. Thesame decision process can be obtained for the Gonly and the G ? A reaction lanes. Sequencescan be confirmed by running replicate reactionson the same gel and comparing the autoradio-graphic patterns between replicates.
Fig. 2.1 The readingpattern of autoradiogram
2 DNA Sequencing: Methods and Applications 13
2.3.2 Sanger Method
Frederick Sanger developed an alternativemethod, rather than using chemical cleavagereactions, Sanger opted for a method involving athird form of the ribose sugar (Sanger et al.1977). Ribose has a hydroxyl group on both the20 and the 30 carbons, whereas deoxyribose hasonly the one hydroxyl group on the 30 carbon.There is a third form of ribose, dideoxyribose inwhich the hydroxyl group is missing from boththe 20 and the 30 carbons (Fig. 2.2). Whenever adideoxynucleotide incorporated into a polynu-cleotide, the chain irreversibly stops or termi-nates. The basic idea behind chain terminationmethod developed in 1974 by Sanger was togenerate all possible single-stranded DNA mol-ecules complementary to a template that starts ata common 50 base and extends up to 1 kilobasein the 30 direction (Fig. 2.3). These single
strands of DNA are labeled in such a way whichallows the identity of the 30-end base in eachmolecule. These molecules are separatedaccording to size by electrophoresis and eachband corresponding to a class of molecule dif-fering in length by one nucleotide from theadjacent band (Fig. 2.4a, b).
2.3.3 Automated DNA SequencingMethods
The principle of automated DNA sequencing issame as Sanger’s method but the detection isdifferent. In this automated method, the primeror the ddNTPs are labeled by incorporation of afluorescent dye. Thus, rather than running thegel for a particular time and reading the results,the machine uses a laser to read the fluorescenceof the dye as the bands pass a fixed point.Labeling of the ddNTPs is much more advan-tageous than the primer labeling because fourddNTPs each labeled with different dyes leadsthe sequencing reaction to run in a single tubeand separated in a single lane, thus increasingthe capacity of the machine. Automated DNAsequencers can sequence up to 384 DNA sam-ples in a single batch and run up to 24 runs perday. DNA sequencers carry out capillary elec-trophoresis for size separation, detection andrecording of dye fluorescence, and data output asfluorescent peak trace chromatograms. Since thecapillary tubes have a high surface to volumeratio (25–100 mm diameter), it radiates heatreadily, thus the samples do not over heat.Detection of the migrating molecules is
Fig. 2.3 Principle of Sanger sequencing
Normal dNTP ddNTP
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Fig. 2.2 Structural comparison of dNTP and ddNTP
14 S. S. Bisht and A. K. Panda
accomplished by shining a light source through aportion of the tubing and detecting the lightemitted from the other side (Fig. 2.5). In thermocycling sequencing the reactions by thermocycling, cleanup, and re-suspension in a buffersolution before loading onto the sequencer areperformed separately. A number of commercialand non-commercial software packages can trimlow-quality DNA traces automatically. Theseprograms score the quality of each peak and
remove low-quality base peaks (generally loca-ted at the ends of the sequence).
2.3.3.1 Base CallingThe raw sequence traces in automatedsequencing can be read using automated soft-wares like Phred programme which converttraces into sequences that can be deposited in adatabase within seconds after sequencing run(Ewing et al. 1998).
A C G T
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Fig. 2.4 Determination of DNA sequence by Sanger’s dideoxy nucleotide method. a Depicted diagram.b Autoradiogram
2 DNA Sequencing: Methods and Applications 15
The new techniques and equipment includedin automated DNA sequencing are:1. Four-color fluorescent dyes have replaced the
radioactive label. Attachment of these dyes tothe ddNTPs results in a fluorescent tag directlymarking just the terminated DNA molecule,and consequently a single sequencing reactionspiked with all four ddNTPs is sufficient tosequence any template.
2. Rather than stopping the electrophoresis at aparticular time the products are scanned forlaser-induced fluorescence just before theyrun off the end of the electrophoresis med-ium. The sequence is collected as a set of four‘‘trace files’’ that indicates the intensity of thefour colors, a peak in the trace distributionimplies that the particular base was the lastone incorporated at the position.
3. Improvement in the chemistry of templatepurification and the sequencing reactionincluding use of bioengineered thermostablepolymerases that can read through secondarystructure with high fidelity extends the lengthof high quality sequence.
4. Slab gel electrophoresis gave way to capil-lary electrophoresis with the introduction in1999 of Applied Biosystem’s ABI Prism3,700 automated sequencers. These se-quencers give extremely high quality, longreads, save time and money by abolishing thelaborious, and often frustrating step of gelpouring that add a new level of automation inwhich the capillaries are loaded by robot
from 96-well plates rather than by hand. Eachmachine can handle six 96-well plates perday or approximately 0.5 Mb of sequence.
5. Matrix-assisted laser desorption/ionization,time-of-flight mass spectrometry (MALDI-TOF MS) was put forward as an alternative tothe Sanger sequencing/capillary electropho-resis combination. It is the tool of choice inproteomics applications, while the fullpotential for DNA analysis was demonstratedin 1995 and for RNA in 1998. For MALDI-TOF MS analysis single-stranded nucleic acidmolecules of 3–29 bp in length(1,000–8,600 Da range) need to be generatedand deposited on a matrix (e.g., 3-hydroxypicolinic acid). The analyte/matrix moleculesare then irradiated by a laser inducing theirdesorption and ionization, upon which themolecules pass through a flight tube con-nected to a detector on the other end. Sepa-ration occurs by the time of flight, which isproportional to the mass of the individualmolecules. The main advantage of the methodis that it directly measures an intrinsic phys-ical property of the molecules i.e., mass andspeed. Limitations lie in the size of the DNAmolecules that can be detected intact to lessthan 100 bp (due to size-dependent fragmen-tation during the MALDI process); and thatthe analytes must be free from ion adductswhich lead to mass distortion.Compared to gel electrophoresis basedsequencing systems, mass spectrometry
TCATTGA
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Migration of DNA fragments
Laser
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Fig. 2.5 Capillary electrophoresis and electropherogram with peaks representing the bands on the sequencing gel
16 S. S. Bisht and A. K. Panda
produces very high resolution of sequencingfragments, rapid separation on microsecondtime scales, and completely eliminates com-pressions associated with gel-based systems.While most of the research efforts have beenfocused on using mass spectrometers to analyzethe DNA products from Sanger sequencing orenzymatic digestion reactions, the read lengthsattainable are currently insufficient for large-scale de novo sequencing. The advantage ofmass-spectrometry sequencing is that one canunambiguously identify frame shift mutationsand heterozygous mutations making it an idealchoice for resequencing projects. In theseapplications, DNA sequencing fragments thatare of the same length but with different basecompositions are generated, which are chal-lenging to consistently distinguish in gel-basedsequencing systems. In contrast, MALDI-TOFMS produces mass spectra of these DNAsequencing fragments with nearly digital reso-lution, allowing accurate determination of themixed bases. For these reasons, mass spec-trometry based sequencing has mainly beenfocused on the detection of frameshift muta-tions and single nucleotide polymorphisms(SNPs). More recently, assays have beendeveloped to indirectly sequence DNA by firstconverting it into RNA. These assays takeadvantage of the increased resolution anddetection ability of MALDI-TOF MS for RNA.
6. For long oligonucleotides ([50 bases), e.g.,microarray applications, Electrospray Ioni-zation-Mass Spectrometry (ESI-MS) is used.The target molecules are ionized into multiplecharge states producing a waveform that canbe de-convoluted into parent peaks. As onlythe charge state will vary for the ions, oligo-nucleotides with high molecular weights canbe analyzed using this method (Edwards et al.2005). In addition, the inherently milder ion-ization conditions make this analytical tech-nique a great tool for the analysis of labilecompounds such as common quenchers, e.g.,dabcyl, BHQ’s, used in dual-labeled fluoro-genic probes. The ESI-MS systems have massresolution of approximately 0.03 %, i.e., res-olution of ±3 Da on a 10 kDa oligonucleotide(Dale and Schantz 2007).
2.4 Genome Sequencing
The genome sequencing usually deals withlarge-scale sequencing, e.g., whole chromo-somes, very long DNA pieces, etc. For longertargets, such as chromosomes, commonapproaches consisting of cutting (with restric-tion enzymes) and shearing (with mechanicalforces) the large DNA fragments into shorterDNA fragments are used. The fragmented DNAis cloned into a DNA vector and amplified inE. coli or other suitable organisms. Short DNAfragments purified from individual clones andsequenced individually called shotgunsequencing, followed by electronic assemblyinto one long contiguous sequence. The over-lapping fragments are joined together to form acontig; two or more contigs assembled to makedraft sequence. This stage contains gaps in theassembled sequence which can be filled byprimer walking and nested deletion strategies.The next stage is the finishing process whichinvolves filling in the gaps and correcting themore obvious errors and uncertainties. The fin-ished sequence does not contain gaps and isaccurate to a defined level. The final stage isannotation which identifies the protein codingsequence. The Human genome project wascompleted by implementing two approaches:clone-by-clone sequencing and whole genomeshotgun sequencing.
2.4.1 Clone-by-Clone Sequencing
In this approach the chromosomes were map-ped and then split up into sections. A roughmap was drawn for each section, and then thesections themselves were split into smaller bits,with plenty of overlap between each of the bits.Each of these smaller bits would be sequenced,and the overlapping bits would be used to putthe genome back together again. First, bymapping the genome researchers produce at anearly stage, a genetic resource that can be usedto map genes. In addition, since every DNAsequence is derived from a known region, it was
2 DNA Sequencing: Methods and Applications 17
relatively easy to keep track of the project and todetermine where gaps are in the sequence.Assembly of relatively short regions of DNA isan efficient step. However, mapping can be atime-consuming and costly process.
2.4.2 Whole Genome ShotgunSequencing
The alternative to the clone-by-clone approach isthe ‘bottom-up’ whole genome shotgun (WGS)sequencing. It was developed by Fred Sanger in1982. First, DNA is broken into fragments fol-lowed by sequencing at random and assemblingtogether the overlaps. Advantage of the wholegenome shotgun is that it requires no priormapping. Its disadvantage is that large genomesneed computing power and sophisticated soft-ware to reassemble the genome from its frag-ments. Unlike the clone-by-clone approach,assemblies cannot be produced until the end ofthe project. Whole genome shotgun for largegenomes is especially valuable if there is anexisting ‘scaffold’ of organized sequences,localized to the genome, derived from otherprojects. When the whole genome shotgun dataare laid on the ‘scaffold’ sequence, it is easier toresolve ambiguities. Today, whole genomeshotgun is used for most bacterial genomes andas a ‘top-up’ of sequence data for many othergenome projects.
2.5 Recent Advances in DNASequencing
During last 5 years many techniques, serviceagencies, and companies have come up scien-tifically with more advanced techniques andcommercially viable services such as 454 lifeSciences, Roche Applied Science, MicrochipBiotechnologies, Agencourt Bioscience, andmany more. The decade after the completion ofthe Human Genome Project, remarkablesequencing technology explosion has permitted amultitude of questions about the genome to beasked and answered at unprecedented speed and
resolution. The advances in sequencing technol-ogy resulted to lower the DNA sequencing costto become negligible for some applications andled far greater range of scientific experiments tobe carried out, but also allow nonsensical uses ofDNA sequencing and generate additional pres-sure on storage resources (Pettersson et al. 2009).
The prominent methods that are receivingsufficient attention from the researchers andscientists working on genome sequencing andrelated research areas of biomedical research aregiven below:1. Illumina sequencing.2. Roche 454 Genome Sequencing.3. Pyro sequencing.4. Solid sequencing.
Apart from these four sequencing methodsfew more are also in practice for varioussequencing studies like Ion semiconductorsequencing, PacBio RS, DNA nanoballsequencing, Lynx Therapeutics’ massively par-allel signature sequencing (MPSS), Polonysequencing (Porreca 2010) etc.
2.5.1 Next-Generation DNASequencing
A new generation of non-Sanger sequencingtechnologies, i.e., next-generation DNAsequencing has the potential to dramaticallyaccelerate biological and biomedical research byenabling the DNA sequencing at unprecedentedspeed. Comprehensive analysis of genomes,transcriptomes, and interactomes has come upwith many advantages like being inexpensive,routine, and widespread rather than requiringsignificant production-scale efforts (Schuster2008; Xiaoguang et al. 2010). First, DNA tem-plate library is constructed. DNA library frag-ments are prepared from either randomlysheared genomic DNA (10 s to 100 s bp in size)or alternatively pair-end fragments with con-trolled distance distribution. The double-strandedfragments are ligated with adaptor sequences atboth ends and denatured. The resulting single-stranded template library is created and
18 S. S. Bisht and A. K. Panda
immobilized on a solid surface (either a planarsurface or supporting beads) and clonallyamplified by one of several means, e.g., bridgePCR, emulsion PCR, or in situ polonies. DNAclusters or amplified beads form an array ofDNA clusters on a slide, which then undergocyclic manipulation through enzyme such aspolymerase or ligase. Optical events generatedfrom the cyclic chain extension process aremonitored by microscopic detection system,and images recorded through CCD camera.Sequential analysis of array image yields DNAfragment sequences, which are assembled intolarger sequence contigs by computer algo-rithm. A comparison of the specifications ofnext generation sequencing technologies isgiven in Table 2.1.
2.5.1.1 Illumina Genome AnalyzerThe amplification of single-stranded libraryfragments is carried out through a processcalled ‘‘bridge amplification.’’ In Illumina/Solexa sequencing [50 million clusters/flowcells, each with 1,000 copies of the sametemplate, 1 billion bases per run, and 1 % ofthe cost of capillary-based method makes itcost effective, highly accurate, straightforwardsample preparation, well established opensource software community and less timeconsuming method. Illumina Hi-seq producesapproximately 1.6 billion short reads(18–150bp) per flow cell, whereas IlluminaMi-seq produces 7.5 Gbase/run producing2 9 250 bp fragments.
The steps are as follows:1 Prepare genomic DNA.2 Attach DNA to surface.3 Bridge amplification.4 Fragment becomes double stranded.5 Denature the double stranded molecules.6 Complete amplification.7 Determine first base.8 Image first base.9 Determine second base.
10 Image second base.11 Sequence reads over multiple cycles.12 Align data. T
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2 DNA Sequencing: Methods and Applications 19
2.5.1.2 Roche 454 Genome SequencerThe 454 Sequencer utilizes emulsion PCR toyield amplicons used for the sequencing proce-dure. Tiny paramagnetic beads coated with DNAprimers are mixed with single-stranded templateDNA library together with components neces-sary for PCR reaction. Proportional amount ofbeads and library fragments are mixed to ensuremost beads carry not more than one ssDNAmolecule. The aqueous solution is mixed withoil to form emulsion where each water com-partment forms an independent micro-reactor forsubsequent PCR chemistry. After multiplerounds of thermo-cycling, each bead is coatedwith thousands of copies of DNA of the samesequence. Beads are further enriched, trans-ferred, and deposited on a picotiter plate fabri-cated in organized array of tiny wells with eachhole occupied by only one bead. The picotiterplate is engineered as part of flow cell forsequencing chemistry on one side and boundedwith optic fibers as part of CCD-based opticaldetection system on the other.
2.5.1.3 PyrosequencingIt is a method of DNA sequencing (determiningthe order of nucleotides in DNA) based on the‘‘sequencing by synthesis’’ principle. It differsfrom Sanger sequencing, in that it relies on the
detection of pyrophosphate release on nucleotideincorporation, rather than chain termination withdideoxynucleotides. The pyrosequencing usuallyrelies on enzymes ATP sulfurylase and lucifer-ase. Release of pyrophosphate, during nucleotidetriphosphate incorporation into the DNA chain,
triggers a cascade of biochemical reaction viaATP sulfurylase and luciferase, resulting in aburst of biochemiluminescent light being emit-ted. Apyrase, a nucleotide degrading enzyme,degrades unincorporated nucleotides, and ATP.After degradation another nucleotide is added.Sequencing is achieved by sequentially intro-ducing each of the four dNTPs into the flow cell.Presence or absence of light burst of each pi-cotiter well indicates the incorporation of corre-sponding nucleotide, therefore, reveals theidentity of complementary base on the templateDNA in that well (Fig. 2.6). Major advantages ofpyrosequencing are its speed and read length-upto 500 bp. Unlike other next-generation tech-nologies discussed, pyrosequencing does notneed to carry out extra chemistries to theextending DNA chain beyond normal biochem-ical process by DNA polymerase, e.g., no need ofremoving label moiety or de-block terminators.
A limitation of the method is that the lengthsof individual reads of DNA sequence areapproximately 300–500 nucleotides, shorter thanthe 800–1,000 obtained with chain terminationmethods (e.g., Sanger sequencing method). Thiscan make the process more difficult, particularlyfor sequences which contain large amount ofrepetitive DNA.
The reactions involved in pyrosequencingare:
NAð ÞnþNucleotide �!Polymerase
NAð Þnþ1þPPi
PPiþ APS �!ATP sulfurylase
ATPþ SO42�
ATPþ Luciferaseþ O2 �!LuciferaseAMPþ PPIþ Oxyluciferase þ CO2 þ Light
2.5.1.4 Solid Sequencing ChemistryLike 454, SOLiD system also employs emulsionPCR as a DNA template amplification scheme withparamagnetic beads. After breaking the emulsion,amplified beads are collected, enriched, and fixedon a flat glass substrate to create a disorder array.
20 S. S. Bisht and A. K. Panda
Its sequencing-by-synthesis is driven by ligationrather than polymerization. Primers hybridize tothe P1 adapter sequence on the templated beads. Aset of four fluorescently labeled di-base probescompete for ligation to the sequencing primer.Specificity of the di-base probe is achieved byinterrogating every first and second base in eachligation reaction. Multiple cycles of ligation,detection, and cleavage are performed with thenumber of cycles determining the eventual readlength. Following a series of ligation cycles, theextension product is removed and the template isreset with a primer complementary to the n-1position for a second round of ligation cycles.
2.5.1.5 Error limitationsThe 454 genome sequencer delivers the longestread length with lowest throughput (8 Mb/hduring a 9-h run) and leads to errors in homo-polymeric tracts, complex sample preparation
and is relatively expensive). Ion torrentsequencer has 454-like chemistry without dyelabeled nucleotides. It can read up to 400 bpreads (single end). In Ion torrent the output isdependent on chip-type. 318 chip producesabout 1 G base in 3 hours. Ion Torrent runproduces the shortest reads and performs poorlywith homopolymers. However, it delivers thefastest throughput (80–100 Mb/h) with shortestrun time (*3 h). Illumina genome analyzercannot read missed base, has lowest rate of basesubstitution errors, difficult to resolve specificmotifs (GGC motifs, inverted repeats) and useslongest-running time (Nakamura et al. 2011).
2.5.1.6 Quality and quantityrequirements in sequencing
Illumine HiSeq lane typically produces about150 million paired end reads under current run
Sulfurylase
APS +PPi ATP
Luciferin Oxyluciferin
Luciferase
ATP Light Pyrogram (light v/s time)
dNTP Apyrase dNDP + dNMP + phosphate
ATP Apyrase ADP + AMP + phosphate
Nucleotide incorporation generates light which is seen as a peak in the program trace
Fig. 2.6 Pyrosequencing
2 DNA Sequencing: Methods and Applications 21
conditions (Haas et al. 2012). Thus, multiplexing15–30 samples per lane will yield the 5–10million reads per sample and will be sufficientfor most applications of bacterial RNA-Seq butstudies of differential gene expression need evensignificantly higher levels of multiplexing result.Approximately 12 9 104 transcripts will becovered by 45 million reads, for low abundancetranscripts high coverage will be required thatdepends on whether it is a low or high abun-dance transcript.
As per ENCODE guidelines, with a referencegenome 20 million reads per sample and morethan 100 million reads per sample without areference are required. Bacterial sample needs 2to 10 million reads per sample.
2.6 Applications of DNASequencing
The Genome sequencing has revolutionized theunderstanding of treatment and prevention ofhuman diseases at affordable price; genomesequencing has got many more significantapplications (Drmanac et al. 2010).
1. DNA sequencing plays vital role in the fieldof agriculture. The mapping and sequencingof the whole genome of microorganisms hasallowed the agriculturists to make themuseful for the crops and food plants. Forexample, specific genes of bacteria havebeen used in some food plants to increasetheir resistance against insects and pests, asa result the productivity and nutritionalvalue of the plants may increase.
2. In medical research, DNA sequencing can beused to detect the genes which are associatedwith some hereditary or acquired diseases.
3. In forensic science, DNA sequencing is usedto identify the criminals by finding someproof from the criminal scene in the form ofhair, nail, skin, or blood samples. DNAsequencing can also be used to determinethe paternity of the child.
4. DNA sequencing information is importantfor planning the procedure and method ofgene manipulation.
5. It is used for construction of restrictionendonuclease maps.
6. It is used to find tandem repeats or invertedrepeat for the possibility of hairpinformations.
7. The sequences can be used to find whetherany open reading frame (ORF) coding for apolypeptide exists or not.
8. DNA sequences can be used to find apolypeptide sequence from the data bank orto compare with DNA sequences from otherorganisms for phylogenetic analysis.
9. DNA sequencing is used to construct themolecular evolution map.
10. Last but not least, it is useful in identifyingexons and introns.
11. DNA sequencing discovers intra and interspecies variations.
12. It can characterize the transcriptome of acell.
13. It can identify DNA bindinig sites forproteins.
14. Metagenomic applications: It uses wholesample DNA/RNA with wide range ofapplications i.e phylogenetics/comparative/functional analysis in addition to environ-mental profiling.
2.7 Conclusion
DNA sequencing is a procedure for identifyingthe base sequence of a fragment of DNA. Themost commonly used procedure is the Sangermethod of DNA sequencing, which uses DNApolymerase I and a PCR reaction to repeatedlysynthesize a new DNA strand using a segment ofDNA as a template. Strand synthesis terminateswhen a dideoxynucleotide is incorporated insteadof a normal nucleotide. Sanger sequencing can bedone manually or by using an automatedsequencer. In manual sequencing, four separatesequencing reactions are performed, one for eachof the four bases. This creates a number of DNAfragments of different lengths which are electro-phoresed on a sequencing gel, fragments differingby a single base in length will separate from eachother and the base sequence of the DNA fragment
22 S. S. Bisht and A. K. Panda
can be determined. An automated sequencer usesa single reaction mixture in which each of the fourterminating dideoxynucleoside triphosphates islabeled with a different fluorescent dye. Thereaction is loaded in a single lane of a sequencinggel and a laser reads the dye color terminatingeach fragment band. Recently, a number of newDNA sequencing methods/sequencers have beendeveloped which are fully automated requiringless time and money, e.g., Illumina GenomeAnalyzer, pyrosquencing, SOLiD SequencingChemistry, 454 Genome Sequencer, etc. DNA/genome sequencing has been applied to forensics,medicine, agriculture, and can be used to detectgenes associated with heredity or acquired dis-eases, identification of microorganisms causingdiseases, increase productivity, and resistanceagainst pests in plants, etc.
References
Dale JW, Schantz MV (2007) DNA sequencing. In: fromgenes to genomes concepts and applications of DNAtechnology, 2nd edn. Wiley Ltd, West Sussex,England
Drmanac R et al (2010) Human genome sequencingusing unchained base reads in self-assembling DNAnanoarrays. Science 327(5961):78–81. doi:10.1126/science.1181498
Edwards JR, Ruparel H, Ju J (2005) Mass-spectrometryDNA sequencing. Mutat Res 573(1–2):3–12. doi:org/10.1016/j.mrfmmm.2004.07.021
Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer races using Phred.I. Accuracy assessment. Genome Res 8:175–185. doi:10.1101/gr.8.3.175
Haas BJ, Chin M, Nusbaum C, Birren BW, Livny J(2012) How deep is deep enough for RNA-Seqprofiling of bacterial transcriptomes? BMC Genomics13: 734
Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, LawM (2012) Comparison of Next-Generation Sequenc-ing Systems. J Biomed Biotechnol 2012:1–11. doi:10.1155/2012/251364
Loman NJ, Misra RV, Dallman TJ, Constantinidou C,Gharbia SE, Wain J, Pallen MJ (2012) Performancecomparison of benchtop high-throughput sequencingplatforms. Nat Biotechnol 30: 434–439. doi:10.1038/nbt.2198
Maxam AM, Gilbert W (1977) A new method forsequencing DNA. Proc Nat Acad Sci 74(2):560–564.PMCID: PMC392330. doi:10.1073/pnas.74.2.560
Nakamura K, Oshima T, Morimoto T, Ikeda S, Yoshik-awa H, Shiwa Y, Ishikawa S, Linak MC, Hirai A,Takahashi H, Altaf-Ul-Amin M, Ogasawara N,Kanaya S (2011) Sequence-specific error profile ofIllumina sequencers. Nucleic Acids Res 39(13). doi:10.1093/nar/gkr34410.1093/nar/gkr344
Pettersson E, Lundeberg J, Ahmadian A (2009) Gener-ations of sequencing technologies. Genomics93(2):105–11. doi.org/10.1016/j.ygeno.2008.10.003
Porreca JG (2010) Genome sequencing on nanoballs. NatBiotechnol 28(1):43–44. doi:10.1038/nbt0110-43
Quail MA, Smith M, Coupland P, Otto TD, Harris SR,Connor TR, Bertoni A, Swerdlow HP, Gu Y (2012) Atale of three next generation sequencing platforms:comparison of Ion Torrent, Pacific Biosciences andIllumina MiSeq sequencers. BMC Genomics 13:341
Sanger F, Nicklen S, Coulson AR (1977) DNA sequenc-ing with chain-terminating inhibitors. Proc Nat AcadSci 74(12):54635467. doi:10.1073/pnas.74.12.5463
Schuster SC (2008) Next-generation sequencing trans-forms today’s biology. Nat Methods 5(1):16–18. doi:10.1038/nmeth1156
Xiaoguang Z, Lufeng R, Yuntao L, Meng Z, Yude Y, JunY (2010) The next-generation sequencing technology:a technology review and future perspective. Sci ChinaLife Sci 53(1):44–57. doi:10.1007/s11427-010-0023-6
2 DNA Sequencing: Methods and Applications 23
3Molecular Markers
Pavan Kumar Agrawal and Rahul Shrivastava
Abstract
Detection and analysis of genetic variation help in understanding themolecular basis of various biological phenomena in eukaryotes. Since theentire eukaryotes cannot be covered under sequencing projects, molec-ular markers and their correlation to phenotypes provide with requisitelandmarks for elucidation of genetic variation. There are different typesof DNA-based molecular markers. These DNA-based markers aredifferentiated into two types; first nonPCR-based (RFLP) and second isPCR-based markers (RAPD, AFLP, SSR, SNP etc.). Amongst others, themicrosatellite DNA marker has been the most widely used in ecological,evolutionary, taxonomical, phylogenetic, and genetic studies due to itseasy use by simple PCR, followed by a denaturing gel electrophoresis forallele size determination and high degree of information provided by itslarge number of alleles per locus. Despite this, a new marker type, namedSNP, for Single Nucleotide Polymorphism, is now on the scene and hasgained high popularity, even though it is only a bi-allelic type of marker.Day by day development of such new and specific types of markersmakes their importance in understanding the genomic variability and thediversity between the same as well as different species of the plants. Inthis chapter, we have discussed types of molecular markers, theiradvantages, disadvantages, and the applications.
3.1 Introduction
The concept of genetic markers is not a new one;Gregor Mendel used phenotype-based geneticmarkers in his experiment in the nineteenthcentury. Later, phenotype-based genetic markersfor Drosophila led to the establishment of thetheory of genetic linkage. The limitations ofphenotype-based genetic markers directed thedevelopment of more general and useful direct
P. K. AgrawalGovind Ballabh Pant Engineering College,Ghurdauri, Pauri, Uttrakhand 246194, Indiae-mail: [email protected]
R. Shrivastava (&)Department of Biotechnology and Bioinformatics,Jaypee University of Information Technology,Waknaghat, Solan, Himachal Pradesh, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_3, � Springer India 2014
25
DNA-based markers that became known asmolecular markers. A molecular marker isdefined as a particular segment of DNA that isrepresentative of the differences at the genomelevel. They may or may not correlate with phe-notypic expression of a trait. Molecular markersoffer numerous advantages over conventionalphenotype-based alternatives as they are stableand detectable in all tissues regardless ofgrowth, differentiation, development, or defensestatus of the cell. Further, these are not con-founded by the environment, pleiotropic andepistatic effects.
Due to the rapid developments in the field ofmolecular genetics, a variety of techniques haveemerged to analyze genetic variation during the
last few decades. These genetic markers maydiffer with respect to important features, such asgenomic abundance, level of polymorphismdetected, locus specificity, reproducibility,technical requirements, and financial invest-ment. No marker is superior to all others for awide range of applications. The most appropri-ate genetic marker depends on the specificapplication, the presumed level of polymor-phism, the presence of sufficient technicalfacilities and knowhow, time constraints, andfinancial limitations. The classifications ofmolecular marker technologies that have beenwidely applied during the last decades aresummarized in Table 3.1.
Table 3.1 Some popular molecular markers, their characteristics and potential applications
Techniques Marker type Requirepriormolecularinformation
Mode ofinheritance
Degree ofpolymorphism
Majorapplications
Discoverer
Non PCR-basedtechniques
Restrictionfragmentlengthpolymorphism
Yes Mendelian,codominant
Low Linkagemapping,Physicalmapping
Bosteinet al.(1980)
PCR-basedtechniques
RandomamplifiedpolymorphicDNA
No Mendelian,dominant
Intermediate Fingerprintingfor populationstudy, Genetagging,Hybrididentification
Williamset al.(1990)
Amplifiedfragmentlengthpolymorphism
No Mendelian,dominant
High Linkagemapping, Genetagging,populationstudy
Vos et al.(1995)
Microsatellite,SSR
Yes Mendelian,codominant
High Linkagemapping,populationstudy, Geneticdiversity,Paternityanalysis
Litt andLutty(1989)
Minisatellite,VNTR
No Mendelian,codominant
High DNAfingerprintingfor populationstudy
Jeffrey(1985)
Singlenucleotidepolymorphism
Yes Mendelian,codominant
High Linkagemapping,populationstudy
Chinget al.(2002)
26 P. K. Agrawal and R. Shrivastava
An ideal molecular marker technique should(1) be polymorphic and evenly distributedthroughout the genome, (2) provide adequateresolution of genetic differences, (3) generatemultiple, independent and reliable markers, (4)be simple, quick, and inexpensive, (5) needsmall amounts of tissue and DNA samples (6)have linkage to distinct phenotypes and (7)require no prior information about the genomeof an organism.
3.2 Molecular Marker Techniques
A vast array of DNA-based genetic markers forthe detection of DNA polymorphism have beendiscovered since 1980 and new marker typesare developed every year. There are severalDNA marker types, which may be classifiedbroadly into two groups based on their detec-tion method, (1) Non PCR based or thosebased on DNA–DNA hybridization and (2)PCR based or those based on amplification ofDNA sequences using the polymerase chainreaction (PCR) (Table 3.1).
3.2.1 Non PCR Based or DNA–DNAHybridization
3.2.1.1 Restriction Fragment LengthPolymorphism
Restriction fragment length polymorphismmarkers were the first DNA-based geneticmarkers developed (Botstein et al. 1980).Eukaryotic genomes are very large and therewas no simple way to observe genetic poly-morphisms of individual genes or sequences.The property of complementary base pairingwhere small piece of DNA could be used asprobe allowed for methods to be developed toreveal polymorphisms in sequences homologousto the probe. The genetic system derived usingthis approach is called restriction fragmentlength polymorphism. RFLP (commonly pro-nounced ‘‘rif-lip’’) refers to a difference betweentwo or more samples of homologous DNA
molecules arising from differing locations ofrestriction sites. If two organisms differ in thedistance between sites of cleavage of particularRestriction endonucleases, the length of thefragments produced will differ when the DNA isdigested with a restriction enzyme. The dissim-ilarity of the patterns generated can be used todifferentiate species (and even strains) from oneanother. In RFLP analysis, the DNA samplebroken into pieces (digested) by restrictionenzymes that are separated according to theirlengths on agarose gel electrophoresis. It ispossible to visualize DNA within such a gel bystaining it with ethidium bromide, however, dueto typically so many restriction fragments of allpossible sizes, discrete fragments cannot beseen. To overcome this problem, the fractionatedDNA is transferred and chemically bound to anylon membrane by a process called Southernblotting, named after its inventor E. M. Southern(1975). Specific DNA fragments are visualizedby hybridizing the DNA fragments bound to thenylon membrane with a radioactively or fluo-rescently labeled DNA probe. Different sizes orlengths of restriction fragments are typicallyproduced when different individuals are tested.Such a polymorphism can be used to distinguishplant species, genotypes and, in some cases,individual plants. Labeling of the probe may beperformed with a radioactive isotope or withalternative non-radioactive stains, such asdigoxigenin or fluorescein. These probes aremostly species-specific single locus probes ofabout 0.5–3.0 kb in size, obtained from a cDNAlibrary or a genomic library. Though genomiclibrary probes may exhibit greater variabilitythan probes from cDNA libraries, a few studiesreveal the converse (Miller and Tanksley 1990).In addition to genetic fingerprinting, RFLP is animportant tool in genome mapping, localizationof genes for genetic disorders, and determinationof risk for disease and paternity testing.
Advantages: The RFLP markers are relativelyhighly polymorphic, codominantly inherited,and highly reproducible. Because of their pres-ence throughout the eukaryotic genome, highheritability and locus specificity of the RFLPmarkers are considered superior. The method
3 Molecular Markers 27
also provides opportunity to simultaneouslyscreen numerous samples. DNA blots can beanalyzed repeatedly by stripping and reprobing(usually 8 to 10 times) with different RFLPprobes.
Disadvantages: The technique is not verywidely used because it is time consuming,expensive, and only one out of several markersmay be polymorphic, which is highly inconve-nient especially for crosses between closelyrelated species. The utility of RFLPs has beenhampered due to the large quantities of purified,high molecular weight genomic DNA require-ment for DNA digestion as well as Southernblotting. The use of radioactive isotope makesthe analysis relatively expensive and hazardous.The requirement of prior sequence informationfor probe generation increases the complexity ofthe methodology. Their inability to detect singlebase changes restricts their use in detecting pointmutations occurring within the regions at whichthey are detecting polymorphism.
Applications: Restriction Fragment lengthpolymorphism is a powerful tool for the identi-fication of organisms to the level of species,strains, varieties, or individuals. RFLPs havebeen widely used in population genetics todetermine slight differences within populations,and in linkage studies to generate maps forquantitative trait locus (QTL) analysis. Theyalso have been used to investigate relationshipsof closely related taxa or intraspecific level, asfingerprinting tools for diversity studies, and forstudies of hybridization. They have been used incriminal and paternity tests, localization ofgenes for genetic disorders, and determination ofrisk for disease.
3.2.2 PCR-Based Marker
After the invention of polymerase chain reaction(PCR) technology (Mullis and Faloona 1987), alarge number of approaches for generation ofmolecular markers based on PCR were detailed,primarily due to its apparent simplicity and highprobability of success. Before PCR, the analysisof a specific DNA fragment generally required
cloning of the fragment and amplification in aplasmid or compatible vector. PCR enables theproduction of a large amount of specific DNAsequence without cloning, starting with just afew molecules of the target sequence. Usage ofrandom primers overcame the limitation of priorsequence knowledge for PCR analysis andfacilitated the development of genetic markersfor a variety of purposes.
3.2.2.1 Amplified Fragment LengthPolymorphism
Amplified fragment length polymorphism PCR(or AFLP-PCR or just AFLP) is a PCR-basedtool used in genetics research, DNA finger-printing and in the practice of genetic engi-neering. Developed by Vos et al. (1995), AFLPis essentially an intermediate between RFLP andPCR. AFLP selectively amplifies a subset ofrestriction fragments from a complex mixture ofDNA fragments obtained after digestion ofgenomic DNA with restriction endonucleases.Polymorphisms are detected from differences inthe length of the amplified fragments by poly-acrylamide gel electrophoresis (PAGE) or bycapillary electrophoresis. AFLP uses restrictionenzymes to cut genomic DNA, followed byligation of adaptors to the sticky ends of therestriction fragments. A subset of the restrictionfragments are then amplified using primerscomplementary to the adaptor and part of therestriction site fragments. The amplified frag-ments are visualized on denaturing polyacryl-amide gels either through autoradiography orfluorescence methodologies (Vos et al. 1995).AFLPs are DNA fragments (80–500 bp)obtained from digestion with restrictionenzymes, followed by ligation of oligonucleo-tide adapters to the digestion products andselective amplification by the PCR. AFLPs,therefore, involve both RFLP and PCR. TheAFLP banding profiles are the result of varia-tions in the restriction sites or in the interveningregion. The AFLP technique simultaneouslygenerates fragments from many genomic sites(usually 50–100 fragments per reaction) that areseparated by polyacrylamide gel electrophoresis
28 P. K. Agrawal and R. Shrivastava
and that are generally scored as dominantmarkers.
Advantages: There are many advantages ofAFLP, they are produced in greater amount andhave higher reproducibility, resolution, andsensitivity at the whole genome level and alsohas the capability to amplify between 50 and 100fragments at one time. In addition, no priorsequence information is needed for amplifica-tion. As a result, AFLP has become extremelybeneficial in the study of taxa including bacteria,fungi, and plants, where much is still unknownabout the genomic makeup of various organ-isms. AFLPs can be analyzed on automatic se-quencers, but software problems concerning thescoring of AFLPs are encountered on somesystems. The use of AFLP in genetic markertechnologies has become the main tool due to itscapability to disclose a high number of poly-morphic markers by single reaction (Vos et al.1995).
Disadvantages: Disadvantages of this tech-nique are that alleles are not easily recognized,have medium reproducibility, labor intensive,and have high operational and developmentalcosts. AFLP needs purified, high molecularweight DNA, the dominance of alleles and thepossible non-homology of co-migrating frag-ments belonging to different loci.
Applications: Most AFLP fragments corre-spond to unique positions on the genome andhave the capability to detect various polymor-phisms in different genomic regions simulta-neously and hence can be exploited aslandmarks in genetic and physical mapping. Thetechnique can be used to distinguish closelyrelated individuals at the subspecies level (Alt-hoff et al. 2007) and also for gene mapping.Applications for AFLP in plant mapping includeestablishing linkage groups in crosses, saturatingregions with markers for gene landing efforts(Yin et al. 1999), and assessing the degree ofrelatedness or variability among cultivars (Mianet al. 2002). Molecular markers are more reliablefor genetic studies than morphological charac-teristics, because the environment does notaffect them. AFLP is considered more applicableto intraspecific than to interspecific studies due
to frequent null alleles. AFLP markers are usefulin genetic studies, such as biodiversity evalua-tion, analysis of germplasm collections, geno-typing of individuals, and genetic distanceanalysis. The availability of many differentrestriction enzymes and corresponding primercombinations provides a great deal of flexibility,enabling the direct manipulation of AFLP frag-ment generation for defined applications (e.g.,polymorphism screening, QTL analysis, andgenetic mapping).
3.2.2.2 Random Amplified PolymorphicDNA
The basis of randomly amplified polymorphicDNA technique is differential PCR amplificationof genomic DNA. It deduces DNA polymor-phisms produced by ‘‘rearrangements or dele-tions at or between oligonucleotide primerbinding sites in the genome’’ using short randomoligonucleotide sequences (mostly 10 baseslong) (Williams et al. 1990). As the approachrequires no prior knowledge of the genome thatis being analyzed, it can be employed acrossspecies using universal primers. In the RAPDmarker system, a PCR reaction is conductedusing a very small amount of template DNA(even less than 10 ng is sufficient) and a singleRAPD primer. Primers are usually just 10 basepairs long (10 mers) and are of randomsequence. The analysis of RAPD is based on thePCR using short (about 10 bases) randomlychosen primers singly which anneal as revertedrepeats to the complementary sites in the gen-ome. The DNA between the two opposite siteswith the primers as starting and end points isamplified by PCR. The amplification productsare separated on agarose gels in the presence ofethidium bromide and viewed under ultravioletlight. The banding patterns distinguish organ-isms according to the presence or absence ofbands (polymorphism). These polymorphismsare considered to be primarily due to variation inthe primer annealing sites, but they can also begenerated by length differences in the amplifiedsequence between primer annealing sites. Eachproduct is derived from a region of the genome
3 Molecular Markers 29
that contains two short segments in invertedorientation, on opposite strands that are com-plementary to the primer. It is a peculiarity ofRAPD analysis that it discriminates at differenttaxonomical level, viz., isolates and species,depending on the organism investigated and theprimer used
Advantages: The main advantage of RAPDsis that they are quick and easy to assay. BecausePCR is involved, only low quantities of templateDNA are required, usually 5–50 ng per reaction.Since random primers are commercially avail-able, no sequence data for primer constructionare needed. Moreover, RAPDs have a very highgenomic abundance and are randomly distrib-uted throughout the genome. They are dominantmarkers; hence have limitations in their use asmarkers for mapping, which can be overcome tosome extent by selecting those markers that arelinked in coupling (Williams et al. 1993). RAPDassay has been used by several groups as effi-cient tools for identification of markers linked toagronomically important traits, which are intro-gressed during the development of near isogeniclines. The RAPD analysis of NILs (nonisogeniclines) has been successful in identifying markerslinked to disease resistance genes in tomato(Lycopersicon sp.), lettuce (Lactuca sp.), andcommon bean (Phaseolus vulgaris). Due to thespeed and efficiency of RAPD analysis, high-density genetic mapping in many plant specieswas developed in a relatively short time.
Disadvantages: The major drawback of themethod is that the profiling is dependent on thereaction conditions so it may vary within twodifferent laboratories and as several discrete lociin the genome are amplified by each primer,profiles are not able to distinguish heterozygousfrom homozygous individuals (Bardakci 2001).
Applications: The application of RAPDs andtheir related modified markers in variabilityanalysis and individual-specific genotyping haslargely been carried out, but is less popular dueto problems such as poor reproducibility, faint orfuzzy products, and difficulty in scoring bands,which lead to inappropriate inferences. RAPDshave been used for many purposes, ranging fromstudies at the individual level (e.g., genetic
identity) to studies involving closely relatedspecies and determination of genetic diversity.RAPDs have also been applied in gene mappingstudies to fill gaps not covered by other markers.
3.2.2.3 Sequence CharacterizedAmplified Region
Sequence characterized amplified regions(SCARs) markers were developed by Michel-more et al. (1991) and Martin et al. (1991).SCARs are DNA fragments amplified by the useof PCR using specific 20–30 bp primers. Theprimers are designed from terminal ends of aRAPD marker. RAPD marker fragments asso-ciated with a phenotypic condition of interest arecloned and sequenced. This nucleotide sequenceis then used for designing of unique primers forspecific amplification of a particular locus. Useof unique primers decreases site competition forspecific regions among primers, making theresults less sensitive to reaction conditions. Useof longer primers also increases the reproduc-ibility of the results by increasing specificity oftemplate binding by the primer. PCR productsobtained after amplification are analyzed for thepresence of length polymorphism by gel elec-trophoresis. Conversion of RAPDs into SCARsmay have additional advantage of obtaining acodominant marker, although dominance may beexhibited by SCARs when one or both primerspartially overlap the site of sequence variation.
Advantages: Due to the application of PCR,low amounts of genomic or template DNA isrequired (10–50 ng per reaction) for analysis ofthe marker. The major advantage of SCARs isthe ease of their use, and requirement of rela-tively less time for the analysis. SCAR markersare locus specific and the results obtained fromanalysis of SCARs are highly reproducible.
Disadvantages: Major limitation lies in thefact that analysis of SCARs can be done only fororganism, species, or DNA fragment withknown sequence; as knowledge of sequence datais required for designing of the PCR primers.
Applications: Major application of SCARmakers has been found in gene mapping studiesas they are locus specific. They are also used in
30 P. K. Agrawal and R. Shrivastava
marker assisted selection, where presence of aspecific genotype and expression of its corre-sponding phenotype can be correlated to thepresence or absence of a SCAR marker (Paranand Michelmore 1993).
3.2.2.4 Minisatellites, Variable Numberof Tandem Repeats
The term ‘minisatellite’ was introduced by Jef-frey et al. (1985). These loci contain tandemrepeats that vary in the number of repeat unitsbetween genotypes and are referred to as vari-able number of tandem repeats (VNTRs) (i.e., asingle locus that contains variable number oftandem repeats between individuals) or hyper-variable regions (HVRs) (i.e., numerous locicontaining tandem repeats within a genomegenerating high levels of polymorphism betweenindividuals). Minisatellites are a conceptuallyvery different class of marker. They consist ofchromosomal regions containing tandem repeatunits of a 10–50 base motif, flanked by con-served DNA restriction sites. A minisatelliteprofile consisting of many bands, usually withina 4–20 kb size range, is generated by usingcommon multi locus probes that are able tohybridize to minisatellite sequences in differentspecies. Locus specific probes can be developedby molecular cloning of DNA restriction frag-ments, subsequent screening with a multi locusminisatellite probe and isolation of specificfragments. Variation in the number of repeatunits, due to unequal crossing over or geneconversion, is considered to be the main cause oflength polymorphisms. Due to the high mutationrate of minisatellites, the level of polymorphismis substantial, generally resulting in uniquemultilocus profiles for different individualswithin a population.
Advantages: The main advantages of mini-satellites are their high level of polymorphismand high reproducibility.
Disadvantages: Disadvantages of minisatel-lites are similar to RFLPs due to the high simi-larity in methodological procedures. Ifmultilocus probes are used, highly informativeprofiles are generally observed due to the
generation of many informative bands perreaction. In that case, band profiles cannot beinterpreted in terms of loci and alleles andsimilar sized fragments may be nonhomologous.In addition, the random distribution of minisat-ellites across the genome has been questioned(Schlötterer 2004).
Applications: The term DNA fingerprintingwas introduced for minisatellites, though DNAfingerprinting is now used in a more general wayto refer to a DNA-based assay to uniquelyidentify individuals. Minisatellites are particu-larly useful in studies involving genetic identity,parentage, clonal growth and structure, andidentification of varieties and cultivars and forpopulation-level studies. Minisatellites are ofreduced value for taxonomic studies because ofhyper variability.
3.2.2.5 Simple Sequence Repeator Microsatellites
The simple sequence repeats (SSR) are alsoreferred to as microsatellites. ‘Microsatellites’,coined by Litt and Lutty (1989), consist of tan-demly repeating units of DNA ubiquitous inprokaryotes and eukaryotes, scattered through-out most eukaryotic genomes (Powell et al.1996). Microsatellites can comprise repetition ofmono-, di-, tri-, tetra- or penta-nucleotide units.The variation in number of repeat units of amicrosatellite resulting in length polymorphismsis mainly attributed to Polymerase slippageduring DNA replication, or slipped strand mi-spairing. Function of such microsatellites islargely unknown, though they can occur inprotein-coding as well as noncoding regions ofthe genome. Microsatellite sequences are espe-cially exploited to differentiate closely relatedspecies or genotypes. Due to the high degree ofvariability present in microsatellites, they arepreferred in population studies (Smith andDevey 1994) and to distinguish closely relatedplant cultivars.
Simple sequence repeat polymorphism isdetected by the use of PCR, in conditions whennucleotide sequences of the flanking regions ofthe microsatellite are known, or by Southern
3 Molecular Markers 31
hybridization using labeled probes. Unlikeminisatellites, which also represent tandemrepeats, microsatellites consist of short tandemrepeat motifs of 1–6 base pair (bp) long units.Knowledge of nucleotide sequence is used todesign specific primers (generally 20–25 bp) foramplification of the microsatellite region alongwith flanking sequences by PCR. Such amplifiedamplicon is then identified by construction of asmall-insert genomic library, followed byscreening the library with a synthetically labeledoligonucleotide probe. Positive clones thusobtained are sequenced for detection and con-firmation of polymorphism.
Microsatellite polymorphism of organismwhose genome is not sequenced may also beidentified by screening sequence databases ofclosely related species for microsatellitesequence motifs. Primers designed from flankingregions after such comparative study are thenused to amplify the SSR region followed byanalysis.
Advantages: High number of alleles and theirco-dominance, along with widespread distribu-tion throughout the genome, are the most sig-nificant features of microsatellites which makethem important molecular markers. The SSRsare present in high genomic abundance ineukaryotic organisms and are mostly localized inlow-copy regions (Morgante et al. 2002). As theanalysis of SSRs is PCR based, low quantities oftemplate DNA (10–50 ng per reaction) arerequired. In comparison to other markers, suchas RAPD, longer primers can be designed andused for microsatellites, thus, increasing repro-ducibility of the results obtained. The analysis ofmicrosatellite can be semi-automated and doesnot necessitate the use of radioactive isotopes.
Microsatellite analysis allows identificationof multiple alleles at a single locus. Although itis done mostly for single locus on the genome, inconditions where the size ranges of the alleles ondifferent loci are markedly different, gel elec-trophoresis analysis or multiplex PCR can easilybe used to simultaneously study multiplemicrosatellites (Ghislain et al. 2004). This wouldhelp in decreasing the analytical costsignificantly.
Individual microsatellite loci can be con-verted into PCR-based markers for analysis asmonolocus, codominant SSRs. In conditionswhere sequence of the microsatellite region isunknown, primers are designed from genomicregions adjacent to the SSR sequences. Theamplified genomic fragment thus obtained iscloned and sequenced to obtain specificsequence of the microsatellite region. Primersare designed using this microsatellite sequencefor specific amplification using PCR. Thisapproach is termed as sequence-tagged micro-satellite site (STMS).
Disadvantages: Principal drawback ofmicrosatellites lies in their high developmentcosts. Sequence information of the template orgenomic DNA is required for designing andsynthesis of primers. Thus, study or analysis ofmicrosatellites is extremely difficult to apply forany unstudied group of species or organism ofinterest. Although SSRs are codominant in nat-ure, mutations in the primer binding sites mayresult in false-negative result because of no-namplification of the intended PCR product(occurrence of null alleles), which may lead toerrors in genotype analysis.
Such null alleles may lead to underestimationof heterozygosity and biased estimate of allelicand genotypic frequencies. Similarly, underes-timation of genetic divergence may occur due tohomoplasy at microsatellite loci, which is aresult of different forward and backwardmutations.
Interpretation of bands obtained on electro-phoretic gel, after PCR amplification of micro-satellite markers may be difficult sometimes.Appearances of artifacts in form of stutter bandshave been seen to influence proper visualizationand size determination of the fragments, thusinterpretation of bands. Formation of such bandsoccurs due to DNA slippage during PCRamplification. Appropriate reference genotypesof known DNA fragments of specific band sizescan be run along with test samples to overcomethe complications in interpretation of suchresults.
Applications: Microsatellites have highdegree of mutability, hence are thought to play a
32 P. K. Agrawal and R. Shrivastava
significant role in genome evolution by creationand maintenance of quantitative genetic varia-tion. Thus, high level of polymorphism shownby microsatellites makes them informativemarkers for population genetics studies, rangingfrom the individual level (e.g., strain identifica-tion) to that of closely related species level.Conversely, due to their high degree of muta-bility, microsatellites markers are not suitablefor analysis and correlation studies of highertaxonomic groups.
SSR markers have been useful for geneticvariation studies in germplasm collections andare considered to be ideal molecular markers forgene mapping studies (Hearne et al. 1992; Jarneand Lagoda 1996). SSRs are molecular markersused for recombination mapping studies, toverify parental relationships and populationgenetic studies. They are the only molecularmarker to provide information about presence orabsence of closely related alleles. Increase ordecrease in the number of SSR repeats in genesof known function can be correlated withchanges in biological functions or phenotypicvariation in the organism (Ayers et al. 1997).SSRs have been shown to be useful for study offunctional diversity among closely related spe-cies, in relation to adaptive changes (Eujay et al.2001).
3.2.2.6 Inter-Simple Sequence RepeatAFLP involves high cost of analysis, whileRAPD is low in reproducibility; analysis of SSRmarkers also requires prior knowledge of DNAsequence of flanking regions for designing ofspecies-specific primers. Thus, analysis ofpolymorphism by application of any of thesetechniques has its limitations, which pose majorobstacle in regular use of these markers. Theselimitations can be overcome to some extent bythe use of ISSR-PCR technique.
SSRs or microsatellites are short segments ofDNA (1–4 base pairs) universally present ineukaryotic genomes. The number of repeats ofSSRs varies in different organisms of same ordifferent species amounting to polymorphism.ISSR is a PCR-based technique, in which SSRs
are used as primers to primarily amplify theinter-SSR regions. The method involves PCRamplification of DNA fragments present at asuitable amplifiable distance in between twoidentical microsatellite regions. The ISSRs areregions between adjacent, oppositely orientedmicrosatellite sequences.
Primers are designed by using microsatelliterepeat regions. As the repeat sequences flankingan inter-SSR region are same, a single primer of17–25 bp length is generally used both as for-ward and reverse primers. The primers used foramplification of ISSRs may consist of di-, tri-,tetra-, or penta-nucleotide repeat units.
The designed primers are usually anchored at30 or 50 end with 1–4 bases extended into thenonrepeat adjacent regions of SSRs (Zietkiewiczet al. 1994). Unanchored primers can also beused for amplification of ISSRs.
Using the primer, about 10–50 inter- SSRsequences of different sizes (about200–3,000 bp) are amplified simultaneouslyfrom multiple genomic loci by a single PCRreaction. These amplicons are separated by gelelectrophoresis and analyzed for the presence orabsence of DNA fragments of particular length.
Some methods related to analysis of ISSRsare:1. Single primer amplification reaction
(SPAR)—that uses a single primer designedonly from the core motif of microsatelliteregion (unanchored primer), and
2. Directed amplification of minisatellite regionDNA (DAMD)—that employs a single pri-mer designed only from the core motif of aminisatellite.Advantages: Principle advantage of ISSRs is
that no sequence data of the fragment to beamplified is required for designing of the prim-ers, as primers are designed from the repeat unitsof flanking microsatellite region. The analysisrequires very small quantities of template DNA(10–50 ng per reaction). ISSRs are mostlyexhibited as dominant marker, though it mightalso be present in codominant state. In compar-ison to RAPD, which is also a PCR-basedtechnique using a single primer, use of longerprimers and corresponding higher annealing
3 Molecular Markers 33
temperature (45–60 �C) leads to higher strin-gency of PCR amplification. Thus, betterreproducibility in results is obtained from anal-ysis of ISSRs as molecular markers.
Disadvantages: As ISSR is a multilocustechnique, fragments with the same size andmobility originating from nonhomologousregions, may lead to misinterpretation of geneticsimilarity estimates. Further, under certain con-ditions ISSRs can also show low reproducibilityof results similar to RAPDs.
Applications: ISSR analysis leads to mul-tilocus fingerprinting profiles, thus it is useful inareas of study of genetic diversity, constructionand study of phylogenetic tree, and evolutionarybiology studies in a wide range of species. ISSRanalysis can be applied in studies involvingparentage establishment, clone and strain iden-tification, and taxonomic studies of closelyrelated species. ISSRs are also considered usefulin genome mapping studies. Though ISSRs arepresent mostly as dominant markers, in somecases they may also segregate as codominantmarkers enabling distinction between homozy-gotes and heterozygotes.
3.2.2.7 Cleaved Amplified PolymorphicSequence
Similar to RFLP, CAPS is based on the principlethat any change or mutation in the genome of anindividual could lead to creation or deletion ofrestriction sites. CAPS markers are generallydeveloped by sequencing of RFLP probes usedfor hybridization. Moreover, 20–25 bp specificprimers are designed for amplification of800–2,000 bp DNA fragments. To increase thechance of finding a region containing polymor-phism introns or 30 untranslated regions aregenerally used for the amplification. Sequencesof DNA from target genotypes are amplifiedusing PCR and the resulting amplicons aredigested individually with one or more restric-tion enzymes. The digested products after gelelectrophoresis show readily distinguishablepattern. Thus, length polymorphisms resultingfrom variation in the occurrence of restrictionsites are identified. CAPS have also been
referred to as PCR-Restriction fragment lengthpolymorphism (PCR–RFLP).
Advantages: Being a PCR-based technique,analysis of CAPS marker requires very lowquantities of template DNA (50–100 ng perreaction). Compared to RFLPs, analysis ofCAPS genotypes does not require the technicallydemanding and time taking steps of Southernhybridization and use of radioactive isotopes.CAPS markers are codominant in nature andlocus specific. Most CAPS markers are easilyscored and interpreted. Results obtained arehighly reproducible.
Disadvantages: Sequence data of the genometo be analyzed are required for synthesis ofprimers. A limited size of DNA fragment can beamplified by PCR reaction, which would be usedfor restriction digestion. Hence, in comparisonto RFLP analysis, specific regions with CAPSpolymorphisms are more difficult to locate.
Applications: CAPS markers have beenapplied predominantly in gene mapping studies,also helpful in identification of mutation in thegenome of an organism.
3.2.2.8 Single NucleotidePolymorphism
A novel class of DNA markers namely singlenucleotide polymorphism in genome (SNPs) hasrecently become highly applicable in genomicstudies. SNP describes polymorphisms causedby point mutations that give rise to differentalleles containing alternative bases at a givennucleotide position within a locus. Suchsequence differences due to base substitutionshave been well characterized since the beginningof DNA sequencing in 1977, but the ability togenotype SNPs rapidly in large numbers ofsamples was not possible until the application ofgene chip technology in the late 1990s. SNPs areagain becoming a focal point in molecularmarker development since they are the mostabundant polymorphism in any organism,adaptable to automation, and reveal hiddenpolymorphism not detected with other markersand methods. Theoretically, a SNP within alocus can produce as many as four alleles, each
34 P. K. Agrawal and R. Shrivastava
containing one of four bases at the SNP site: A,T, C, and G. Practically, however, most SNPsare usually restricted to one of two alleles (mostoften either the two pyrimidines C/T or the twopurines A/G) and have been regarded as bi-allelic.
Analytical procedures require sequenceinformation for the design of allele specific PCRprimers or oligonucleotide probes. SNPs andflanking sequences can be found by libraryconstruction and sequencing or through thescreening of readily available sequence dat-abases. Once the location of SNPs is identifiedand appropriate primers designed, one of theadvantages they offer is the possibility of high-throughput automation. To achieve high samplethroughput, multiplex PCR and hybridization tooligonucleotide microarrays or analysis onautomated sequencers are often used to interro-gate the presence of SNPs. SNP analysis may beuseful for cultivar discrimination in crops whereit is difficult to find polymorphisms, such as inthe cultivated tomato. SNPs may also be used tosaturate linkage maps in order to locate relevanttraits in the genome. For instance, in Arabid-opsis thaliana a high density linkage map foreasy to score DNA markers was lacking untilSNPs became available (Cho et al. 2000; Chinget al. 2002). To date, SNP markers are not yetroutinely applied in gene banks, in particularbecause of the high costs involved.
SNP markers are inherited as codominantmarkers. Several approaches have been used forSNP discovery including single-strand confor-mational polymorphism assays analysis, hetero-duplex analysis, and direct DNA sequencing.DNA sequencing has been the most accurate andmost used approach for SNP discovery. Randomshotgun sequencing, amplicon sequencing usingPCR, and comparative EST analysis are amongthe most popular sequencing methods for SNPdiscovery.
Advantages: Despite technological advances,SNP genotyping is still a challenging endeavorand requires specialized equipment. Traditionalmethods available for SNP genotyping include:direct sequencing, single base sequencing, allele
specific oligonucleotide, denaturing gradient gelelectrophoresis (DGGE), SSCP, and ligationchain reaction (LCR). Each approach has itsadvantages and limitations, but all are still usefulfor SNP genotyping, especially in small labora-tories limited by budget and labor constraints.Large-scale analysis of SNP markers, however,depends on the availability of expensive, cut-ting-edge equipment. Several options are avail-able for efficient genotyping using state-of-the-art equipment.
Particularly, popular are methods involvingmatrix-assisted laser desorption ionization timeof flight (MALDI-TOF) mass spectrometry, py-rosequencing, Taqman allelic discrimination,real-time (quantitative) PCR, and the use ofmicroarray or gene chips. Mass spectrometryand microarray technologies require a largeinvestment in equipment.
Applications: In plants, SNPs are rapidlyreplacing simple sequence repeats as the DNAmarker of choice for applications in plant breed-ing and genetics because they are more abundant,stable, amenable to automation, efficient, andincreasingly cost-effective (Duran et al. 2009).Generally, SNPs are the most abundant form ofgenetic variation in eukaryotic genomes. More-over, they occur in both coding and noncodingregions of nuclear and plastid DNA. As in thecase of human genome, SNP-based resources arebeing developed and made publicly available forbroad application in rice research. These resour-ces include large SNP datasets, tools for identi-fying informative SNPs for targeted applications,and a suite of custom-designed SNP assays foruse in marker assisted and genomic selection.SNPs are widely used in breeding programs forseveral applications such as, (1) marker assistedand genomic selection, (2) association and QTLmapping, positional cloning, (3) haplotype andpedigree analysis, (4) seed purity testing, (5)variety identification, and (6) monitoring thecombinations of alleles that perform well in targetenvironments (Kim et al. 2010).
The advantage and disadvantage of commonmolecular marker techniques are summarized inTable 3.2.
3 Molecular Markers 35
3.3 Array-Based Platforms
Several different types of molecular markers havebeen developed over the past three decades (Guptaand Rustgi 2004), motivated by requirements forincreased throughput, decreased cost per datapoint, and greater map resolution. Recently, oli-gonucleotide-based gene expression microarrayshave been used to identify DNA sequence poly-morphisms using genomic DNA as the target.
3.3.1 Diversity Arrays Technology
Diversity arrays technology (DArT) is micro-array hybridization-based technique that permits
simultaneous screening of thousands of poly-morphic loci without any prior sequence infor-mation. The DArT methodology offers a highmultiplexing level, being able to simultaneouslytype several thousand loci per assay, while beingindependent of sequence information. DArTassays generate whole genome fingerprints byscoring the presence versus absence of DNAfragments in genomic representations generatedfrom genomic DNA samples through the processof complexity reduction. DArT has been devel-oped as a hybridization-based alternative to themajority of gel-based marker technologies cur-rently in use. It can provide hundreds to tens ofthousands of highly reliable markers for anyspecies as it does not require any preciseinformation about the genome sequence
Table 3.2 Advantages and disadvantage of commonly used genetic markers
Marker Advantages Disadvantages
Amplified fragment lengthpolymorphism
Sequence information notrequired
Very tricky due to changes in patterns withrespect to materials used
Can be used across species
Work with smaller RFLPfragments
Low reproducibility
Useful in preparing contig maps Need to have very good primers
High genomic abundance
High polymorphism
Restriction fragment lengthpolymorphism
Sequence information notrequired
Large amount of good quality DNA required
High genomic abundance
Codominant markers
High reproducibility Labour intensive (in comparison to RAPD)
Can use filters many times
Good genome coverage Difficult to automate
Can be used across species Need radioactive labeling
Needed for map-based cloning Cloning and characterization of probe arerequired
Randomly amplifiedpolymorphic DNA
Sequence information notrequired
No probe or primer information
Dominant markers
Very low reproducibility
Good genome coverage Cannot be used across species
Ideal for automation Not well established
Less amount and poor qualityDNA acceptable
No radioactive labeling
Relatively faster
High genomic abundance
36 P. K. Agrawal and R. Shrivastava
(Jaccoud et al. 2001). Moreover, DArT wasfound to provide good genome coverage inwheat and barley (Akbari et al. 2006). Animportant step of this technology is a step called‘‘genome complexity reduction’’ which increa-ses genomic representation by reducing repeti-tive sequences that are abundant in eukaryotes.With DArT platform, comprehensive genomeprofiles are becoming affordable for virtuallyany crop, genome profiles which can be used inmanagement of bio-diversity, for example ingermplasm collections. DArT genome profilesenable breeders to map QTL in 1 week.
DArT profiles accelerate the introgression ofa selected genomic region into an elite geneticbackground (for example, by marker assistedbackcrossing). In addition, DArT profiles can beused to guide the assembly of many differentregions into improved varieties (marker assistedbreeding). The number of markers DArT detectsis determined primarily by the level of DNAsequence variation in the material subjected toanalysis and by the complexity reductionmethod deployed. Another advantage of DArTmarkers is that their sequence is easily accessi-ble compared to amplified fragment lengthpolymorphisms making DArT a method ofchoice for non-model species (James et al.2008). DArT has also been applied to a numberof animal species and microorganisms.
3.3.2 Restriction Site-Associated DNA
Another high-throughput method is restrictionsite-associated DNA (RAD) procedure whichinvolves digestion of DNA with a particularrestriction enzyme, ligating biotinylated adapt-ers to the overhangs, randomly shearing theDNA into fragments much smaller than theaverage distance between restriction sites, andisolating the biotinylated fragments usingstreptavidin beads (Miller et al. 2007a). RADspecifically isolates DNA tags directly flankingthe restriction sites of a particular restrictionenzyme throughout the genome. More recently,the RAD tag isolation procedure has been
modified for use with high-throughput sequenc-ing on the Illumina platform. In addition, Milleret al. (2007b) demonstrated that RAD markers,using microarray platform, allowed high-throughput, high-resolution genotyping in bothmodel and non-model systems.
3.3.3 Single Feature Polymorphism
A third high-throughput method is single featurepolymorphism (SFP) which is done by labelinggenomic DNA (target) and hybridizing toarrayed oligonucleotide probes that are com-plementary to insert-delete (INDEL) loci. TheSFPs can be discovered through sequencealignments or by hybridization of genomic DNAwith whole genome microarrays. Each SFP isscored by the presence or absence of a hybrid-ization signal with its corresponding oligonu-cleotide probe on the array. Both spottedoligonucleotides and Affymetrix-type arrayshave been used in SFP. Borevitz et al. (2003)coined the term ‘‘single feature polymorphism’’and demonstrated that this approach can beapplied to organisms with somewhat largergenomes, specifically A. thaliana with a genomesize of 140 Mb. Similarly, whole genome DNA-based SFP detection has been accomplished inrice (Kumar et al. 2007), with a genome size of440 Mb, barley, which has a 5,300 Mb genomecomposed of more than 90 % repetitive DNA(Cui et al. 2005). Thus, SFPs have become anattractive marker system for various applicationsincluding parental polymorphism discovery. Thedevelopment of DNA-based technologies suchas SFP, DArT, and RAD which are based onmicroarray have the merits of SNP withoutgoing through sequencing. These technologieshave provided us platforms for medium- to ultra-high-throughput genotyping to discover regionsof the genome at a low cost, and have beenshown to be particularly useful for genomes,where the level of polymorphism is low (Guptaet al. 2008). These array-based technologies areexpected to play an important role in cropimprovement and will be used for a variety of
3 Molecular Markers 37
studies including the development of high-den-sity molecular maps, which may then be used forQTL interval mapping and for functional andevolutionary studies.
3.4 Conclusion
DNA-based molecular markers have acted asversatile tools and have found their own positionin various fields like taxonomy, physiology,embryology, genetic engineering, etc. They areno longer looked upon as simple DNA finger-printing markers in variability studies or as mereforensic tools. Ever since their development,they are constantly being modified to enhancetheir utility and to bring about automation in theprocess of genome analysis. The discovery ofPCR was a landmark in this effort and proved tobe a unique process that brought about a newclass of DNA profiling markers. This facilitatedthe development of marker based gene tags,map-based cloning of agronomically importantgenes, variability studies, phylogenetic analysis,synteny mapping, marker assisted selection ofdesirable genotypes, etc. Thus, molecular mar-ker provides new dimensions to concerted effortsof breeding and marker aided selection that canreduce the time span of developing new andbetter varieties and will make the dream of supervarieties come true. These DNA markers offerseveral advantages over traditional phenotypicmarkers, as they provide data that can be ana-lyzed objectively. With the advent of molecularmarkers, it is now possible to make directinferences about genetic diversity and interrela-tionships among organisms at the DNA levelwithout the confounding effects of the environ-ment and/or faulty pedigree records. Geneticanalyses of plant and animal populations andspecies for taxonomic, evolutionary, and eco-logical studies tremendously benefited from thedevelopment of various molecular marker tech-niques. Each molecular marker technique isbased on different principles but their applicationis to bring out the genome-wide variability.
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Yin X, Stam P, Dourleijn CJ, Kropff MJ (1999) AFLPmapping of quantitative trait loci for yield-determin-ing physiological characters in spring barley. TheorAppl Genet 99:244–253
Zietkiewicz E, Rafalski A, Labuda D (1994) Genomefingerprinting by simple sequence repeat (SSR)anchored polymerase chain reaction amplification.Genomics 20:176–183
3 Molecular Markers 39
4Gene Therapy
K. Rohini
Abstract
Genes are the blueprints for proteins and serve as building blocks fortissues. Genes are regulators of chemical reactions inside the living cells.Mutation in a gene causes a change in the expressed structure of proteinleading to protein dysfunction. Genetic errors disrupt gene expressionand result in diseases. Advancement in biotechnology has helped inunderstanding the genetic basis of inherited diseases. Gene therapy is anexperimental technique that uses genes as medicine for correctingdefective genes that are responsible for disease development. Genetherapy has the potential to eliminate a wide range of inherited andacquired human diseases. A number of clinical trials in gene therapy arebeing carried out throughout the world. Gene therapy approach involvesthe treatment of disease by introducing new genetic instructions into thetissues of patients to correct the defective or abnormal gene. In genetherapy, mostly somatic (nonsex) cells are targeted for treatment. Due tocontroversies associated with the involvement of germ cells, this therapyis not attempted. As somatic gene therapy is directed at the individual, ithas no impact on future generations. Changes directed at somatic cellsare not inherited. The problem of ‘‘gene delivery’’ i.e., the need to getreplacement genes into the desired tissues is the most difficult part andneeds intensive research. The majority of current gene therapy clinicaltrials involve the mechanisms of viruses as ‘‘vectors’’. These viral vectorsare capable of integrating at random sites in the host’s cells. To date,gene therapy is being used to treat several genetic disorders. A largenumber of clinical trials are underway to test gene therapy as a treatmentof choice for many life-threatening diseases.
K. Rohini (&)Unit of Biochemistry, Faculty of Medicine, AIMSTUniversity, 3� Bukit Air Nasi, Jalan BedongSemeling, 08100 Bedong, Kedah Darul Aman,Malaysiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_4, � Springer India 2014
41
4.1 Introduction
Genes are hereditary units, carried on a chro-mosome. Mutation of the gene causes a pro-tein dysfunction due to the change in thecodon, which changes the amino acid.Researches in biotechnology are progressingby leaps and bounds and gene therapy is oneof the most rapidly advancing areas ofresearch.
Gene therapy is the use of genes as medi-cines which is an experimental technique forcorrecting defective genes, thus altering thecourse of a medical disease. It primarilyinvolves genetic manipulations in animals orhumans to correct a disease and is applied inseveral fields such as cancer therapy, mono-genic diseases, infectious disease, vasculardiseases, autoimmune diseases etc. Geneticdiseases can be inherited, occur at birth or to berandomly acquired due to environmental factorssuch as exposure to toxins. Single gene disor-ders, such as cystic fibrosis (CF), hemophilia,muscular dystrophy, and sickle cell anemia, arecaused by a single gene malfunctioning and areusually inherited.
In the early 1900s adenosine deaminase(ADA) deficiency, an immuno-deficiency statewas identified and was recorded as the firstcondition to be treated with gene therapy.Therapeutic gene transfer into stem cells in theclinical arena was attempted for the first time forthis condition. A mile stone in medical historywas the successful treatment of another form ofimmune deficiency called severe combinedimmune deficiency (SCID).
Since involvement of gene therapy evokesgreater risk, hence there are several regulatoryagencies whose permission must be soughtbefore any work is initiated. Recombinant DNAAdvisory Committee (RAC) is the supervisorybody of the National Institute of Health,involving gene therapy. Numerous researchesin genetic disorders and other diseases arecurrently at various stages of clinical trials.Some significant examples are listed inTable 4.1.
4.2 Approaches to Gene Therapy
The two main approaches to gene therapy are:
4.2.1 Somatic Gene Therapy
Somatic cells are the nonreproductive cells of anorganism (other than sex cells), e.g., blood cells,bone marrow cells, intestinal cells etc. Somaticcell gene therapy involves the insertion of fullyfunctional and expressible gene into a targetsomatic cell to correct genetic disease. Insomatic gene therapy, the fully functional ther-apeutic gene is inserted into the target somaticcell of a patient to correct the genetic diseasepermanently. Somatic gene therapy will not beinherited by the patient’s offspring or latergenerations and any effects will be restricted tothe individual patient only.
4.2.2 Germ Line Gene Therapy
The reproductive (sex) cells, i.e., sperm or eggsof an organism constitute germ cell line. Germline gene therapy is the introduction of func-tional genes, which are integrated into genomes.Hence, the change due to germ line therapy isheritable and would be passed to later genera-tions. This approach should be highly effectivein treatment of genetic and hereditary disorders,however, for safety, ethical and technical rea-sons, this therapy is generally not attempted.
4.3 Principles of Gene Therapy
Gene therapy is introducing a functional copy ofthe gene for curing the defect. If a child or foetusis diagnosed to carry a defective gene leading todisability, one may like to correct it by one ofthe three methods: (1) by replacing the defectivegene by correct gene, (2) by correcting thedefective gene through gene targeting or (3) bygene augmentation either through increasing thenumber of copies of the gene or through a higher
42 K. Rohini
level of expression of introduced gene. Maxi-mum progress in the area of gene therapy hasbeen achieved through the gene augmentationmethod where normal foreign gene sequencesfor defective gene are introduced involvingvector-mediated DNA delivery system.
The steps involved in the gene therapy are asfollows:1. Identification and characterization of gene.2. Cloning of gene.3. Choice of vector.4. Method of delivery.5. Expression of gene.
The primary step is to identify the geneticdefect and to clone a functional copy of thegene. The functional gene is then transferred tothe patient, which involves choosing a vectorwith a suitable method of delivery. The vector/gene must be designed to allow proper expres-sion of the gene inside the patient. There are twotypes of gene therapies:
4.3.1 Ex Vivo Gene Therapy
Ex vivo gene therapy involves the transfer ofgenes in cultured cells which are then reintro-duced into the patient (therapy takes place out-side the patient). This technique involves thefollowing steps:1. Isolation of cells (selected tissues e.g., bone
marrow) with genetic defect from a patient.2. Growing the cells in culture.3. Introduction of the therapeutic gene to cor-
rect the defective gene.4. Selection of the genetically corrected cells
and their growth.
5. Transplantation of the modified cells to thepatient.This therapy involves the use of patient’s own
cells for culture and genetic correction and thentransferred back to the patient. Hence, thistherapy does not develop adverse immunologi-cal reactions and is quite effective (Fig. 4.1).
4.3.2 In Vivo Gene Therapy
In vivo gene therapy is the direct delivery of thegenes into the cells of the particular tissue.
4.4 Vehicles of Gene Transfer
A gene cannot be directly inserted into a per-son’s cells. It must be delivered to the cell usinga carrier, or ‘‘vector’’ (Table 4.2). Gene trans-ferring systems can be divided into two groups,viral and nonviral vectors. The examples of viralvectors are: (a) Retrovirus, (b) Adenovirus,(c) Adeno-associated virus (AAV), and theexamples of nonviral vectors are: (a) Liposomes,(b) Ribozymes, and (c) Oligonucleotides.
4.4.1 Viral Vectors in Gene Delivery
4.4.1.1 Recombinant Retrovirus VectorsThe most common class of gene transfer vectoris the recombinant retroviruses. Retrovirusescontain single-stranded RNA genome of7–10 kb in size. These genomes are complexedwith vector-encoded proteins and surrounded
Table 4.1 Selected list of some important human gene therapy trials
Disease Gene therapy
Severe combined immunodeficiency (SCID) Adenosine deaminase (ADA)
Cystic fibrosis Cystic fibrosis transmembrane regulator (CFTR)
Sickle cell anemia b-Globin
Hemophilia Factor IX
Thalassemia a or b–Globin
Peripheral artery disease Vascular endothelial growth factor (VEGF)
Familial hyper cholesterolemia Low-density lipoprotein (LDL) receptor
Lesch–Nyhan syndrome Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
4 Gene Therapy 43
with a lipid bilayer generated when the virusbuds from the infected cell. Retrovirus genomesintegrate into the target cell chromosomes asdouble-stranded DNA provirus generated byreverse transcription. This aids the virus gen-omes to be stably transferred to the targetedcells. Examples of retroviruses used as recom-binant vectors include: oncoretroviruses, suchas Moloney Murine Leukemia virus (MuLV)
associated with oncogenesis in mice; lentivi-ruses, such as human immunodeficiency virus(HIV) associated with AIDS in humans; andspumaviruses, such as the human foamy virus,that has no known pathogenesis in humans(Buchschacher 2001; Bushman 2007).
The basic retrovirus genome consists of threegenes (gag, pol and env) enclosed between twolong terminal repeats (LTRs) (Fig. 4.2). The
Fig. 4.1 Strategies for delivering therapeutic transgenes into patients; transfer of genes in cultured cells (ex vivo) anddirect delivery of genes into the cells (in vivo) � 2001 Terese Winslow
Table 4.2 Gene transfer vehicles–vector delivery system include either recombinant viruses or various forms ofplasmid DNA
Source Vector Genome Advantages Disadvantages
Viral Retrovirus ssRNA Efficient transduction Transduction requires cell division
Stable gene transfer Limits on transgene size/content
Relatively safe
Adenovirus dsDNA Highly effective gene transfer Transient transduction
Higher titer Immunological response
Adeno-associatedvirus (AAV)
ssDNA Potential stable transduction Status of genome not fullyelucidatedStable expression
Nonviral Plasmid/Liposomes dsDNA Absence of viral components Low transfer and expression
Potential for targeting
44 K. Rohini
LTR sequences are needed for integration of theDNA version of the virus genome into the hostcell DNA. Between the upstream or 50 LTR andthe gag gene is the packaging signal (psi W)which is essential for packaging the RNA intothe budding virus particle, and the codingsequences for the reverse transcriptase andstructural genes needed for the formation ofmature virus particle.
The first step in generating replication-defec-tive recombinant vectors is to introduce thecoding sequences separately into cultured cells togenerate packaging lines. The therapeutic gene isthen combined with the remaining viral sequen-ces, including the LTR and W elements, in bac-terial plasmids. This plasmid form of a vector isintroduced into the packaging line, where theprimary RNA transcripts are incorporated intothe empty virus particles generated by the viralstructural genes (Fig. 4.3). As a result, these
virus particles contain the recombinant vectorgenomes, but not virus structural genes. Afterinfection of the patient, the RNA inside the ret-roviral vector is reverse transcribed to give aDNA copy (although the retroviral vector doesnot carry a copy of the reverse transcriptase gene,a few molecules of reverse transcriptase enzymeare packaged in retrovirus particles). Ideally, thecloned gene, enclosed between the two LTRsequences, is then integrated into host cell DNA(Wolff and Budker 2005).
Recombinant retro viral vectors are advan-tageous because the integration of vectors intothe target cell chromosomes is highly stableand do not cause an immune response. Thisclass of vectors is also capable of highly effi-cient rates of gene transfer, depending on thechoice of virus envelope. There are also severaldisadvantages for this class of recombinantvirus vectors, which include a variable rate of
LTR gag pol env LTR
Fig. 4.2 Retrovirus genome has packaging signal (W) and gag, pol and env genes
Packaging Construct
Vector genome packaged inside the construct
Retrovirus vector particle
The vector particle is ready for transfer into patient
LTR gag pol env LTR
LTR Cloned gene LTR
Fig. 4.3 Retrovirus vectorsystem uses two virusconstructs. The therapeuticvector carried the clonedgene and packaging signal.When both the constructsare present, the therapeuticvector and the cloned geneis packaged into the capsid(Mandel et al. 2006)
4 Gene Therapy 45
gene expression. They can carry only smallamounts of DNA (about 8 kb) and cannotinfect nondividing cells. These vectors are dif-ficult to deliver in vivo because they are rela-tively fragile. Besides, there are growing safetyconcerns about potential problems of muta-genesis (Weidhaas et al. 2000).
4.4.1.2 Recombinant AdenovirusVectors
Adenoviruses were among the first viruses chosenas vectors for use in human gene therapy andcurrently the second most common form of genetransfer vectors. Adenoviruses are relativelylarge 36 kb linear double-stranded DNA viruses
(a)
(b)
Fig. 4.4 a Adenovirus structure. b Adenovirus entershuman cells by recognizing two receptors CAR andintegrin. The virus is taken into the cell attached to the
receptors and injects its DNA into the nucleus through anuclear pore
46 K. Rohini
that contain many regulatory and structural genesthat are expressed at different times during theinfection (Fig. 4.4a). These genomes are sur-rounded by glycoprotein capsids that are gener-ated through lytic life cycle. The virus particleconsists of a simple icosahedral shell, or capsid,containing a single linear dsDNA molecule ofapproximately 36,000 base pairs. A terminalprotein protects each end of the DNA. The capsidis made of 240 hexons, each of which is a trimerof the hexon protein. Hexons are named for their6-fold symmetry and are surrounded by sixneighboring hexons. The hexon protein has loopsthat project outward from the virus surface.Adenoviruses are subdivided into about 50 sero-types by their response to antibody binding. Thisvariation is largely due to variation in the loops ofthe hexon protein. The adenoviruses share thesame receptor as B-group coxsackieviruses. Thisprotein is therefore known as coxsackievirusadenovirus receptor (or CAR), but its normalphysiological role is unknown (Wu et al. 2006).
Adenoviruses enter human cells by recog-nizing two receptors, CAR and integrin. Thevirus is taken into the cell attached to thereceptors and surrounded by a membrane vesiclethat dissociated in the cytoplasm. The adenovi-rus is injected into the nucleus through a nuclearpore. After the fiber tip binds to CAR, the pentonbase binds to integrins on the host cell surface(integrins are transmembrane proteins involvedin adhesion) (Fig. 4.4b). The membrane puckers,move inward and form a vesicle that takes theadenovirus inside the cell. The virus is thenreleased into the cytoplasm and travels towardthe nucleus. The virion is disassembled outsidethe nucleus, and only the DNA (with its terminalproteins) enters the nucleus.
Adenoviruses have several advantages:1. They are relatively harmless and cause mild
infections of epithelial cells.2. They are nononcogenic (i.e., they do not
cause tumors).3. They are relatively easy to culture and can be
produced in large quantities.4. The life cycle and biology of adenovirus are
well understood.
5. The functions of most adenovirus genes areknown. They include a highly efficienttransfer associated with a large number ofvector copies per cell.However, adenoviruses do cause inflamma-
tion and can cause serious illness in patientswith damaged immune systems. Therefore,when designing an adenovirus vector for genetherapy, the virus needs to be disarmed bycrippling its replication system. This is done bydeleting the gene for E1A protein, a virus pro-tein made immediately on infection. E1A hastwo functions. First, it promotes transcription ofother early virus genes. Second, it binds to hostcell Rb protein, which normally prevents thecell from entering S-phase. This prompts thehost cell to express genes for DNA synthesis,which the virus utilizes for its own replication.In the lab, crippled adenovirus is grown ingenetically modified host cells that have theviral E1A gene integrated into host cell DNA.The virus particles generated by this approachcannot replicate in normal animal cells (StGeorge 2003).
The main drawback of this class of vectorsinvolves the transient nature of gene transfer andexpression, which often lasts only a couple ofmonths in nondividing cells and shorter time individing cells. Several safety concerns are alsoassociated with these vectors. They can havelethal toxicity at high doses and are also highlyinfectious in humans.
4.4.1.3 Recombinant Adeno-AssociatedVirus (AAV) Vectors
AAVs are being used increasingly for clinicalgene transfer and show considerable promise.AAVs are encapsidated parvoviruses with small(5 kb) linear single-stranded DNA genomes thatencode only two gene products. AAV is adefective or ‘‘satellite’’ virus that depends onadenovirus (or some herpes viruses) to supplysome necessary functions. AAV can replicateboth as an episome and as an integrated provi-rus, depending on several factors including celltype.
4 Gene Therapy 47
AAVs are produced by first generating aplasmid form of the vector containing the ther-apeutic gene and critical cis-regulatory elementssuch as the inverted terminal repeats and pack-aging signal. This is combined with helperplasmids that express the AAV coding genes.These plasmids are co-transfected into a pack-aging cell line that contains the critical adeno-virus E1 gene, so that the vector genomes canreplicate. The transfected cells are eventuallylysed and the virus particles are collected andrun over a gradient or some other separationtechnique to purify the virus particles that con-tain only the vector genomes (Miller et al. 2002).
The advantages of using AAV are as follows:1. It does not stimulate inflammation in the host.2. It does not provoke antibody formation and
can therefore be used for multiple treatments.3. It infects a wide range of animals, as long as
an appropriate helper virus is also present,hence can be cultured in many types of ani-mal cells, including those from mice ormonkeys.
4. It can enter nondividing cells of many dif-ferent tissues, unlike adenovirus.
5. The unusually small size of the virus particleallows it to penetrate many tissues of thebody effectively.
6. AAV integrates its DNA into a single site inthe genome of animal cells (the AAV S1siteon chromosome 19 in humans). This allowsthe therapeutic gene to be permanentlyintegrated.The main drawback of this class of vectors is
that they can only accommodate small transg-enes; transfer and expression is not always stabledue to tissue-specific variations.
4.4.2 Nonviral Vectors in GeneDelivery
Although viral vectors are highly sophisticatedand about 75 % of gene therapy trials have usedthese vectors only, nonviral vectors makeattractive vehicle as they are inherently safer. Avariety of alternative approaches investigatedand found widely effective include:
1. Use of naked nucleic acid (DNA or less oftenRNA): Naked DNA is an attractive nonviralvector because of its inherent simplicity as itcan easily be produced in bacteria andmanipulated using standard recombinantDNA techniques. It shows very little dissem-ination and transfection at distant sites fol-lowing delivery and can be re-administeredmultiple times into mammals (including pri-mates) without inducing an antibody responseagainst itself (i.e., no anti-DNA antibodiesgenerated) (Li and Huang 2006). Direct in vivogene transfer with naked DNA was firstdemonstrated when efficient transfection ofmyofibers was observed following injection ofmRNA into skeletal muscle. Subsequentstudies also found foreign gene expressionafter direct injection in other tissues such asheart, thyroid, skin, and liver. Since nakedDNA is the simplest and safest gene deliverysystem, multiple approaches have beenattempted for in vivo liver gene delivery,including intraportal injection, increasedDNA retention time in the liver, electrogenetransfer, hydrodynamic IV injection, andmechanical liver massage. Hydrodynamictransfection had been widely used because ofits simplicity and high efficiency, and althoughnucleic acid delivery has been observed in allmajor organs, the best results are found in theliver (Niidome and Huang 2002).
2. Particle bombardment: In this technique,DNA is fired through the cell walls andmembranes on metal particles. This methodwas originally developed to get DNA intoplants. However, it has also been used tomake transgenic animals and is occasionallyused for humans.
3. Receptor-mediated uptake: DNA in this caseis attached to a protein that is recognized by acell surface receptor. When the protein entersthe cell, the DNA is taken in with it.
4. Polymer-complexed DNA: Binding to a pos-itively charged polymer, such as polyethyl-eneimine, protects the negatively chargedDNA. Such complexes are often taken up bycells in culture and may in principle be usedfor ex vivo gene therapy.
48 K. Rohini
5. Encapsulated cells: Whole cells engineeredto express and secrete a needed protein maybe encapsulated in a porous polymeric coatand injected locally. This approach may be ofgreat value in treating conditions like Alz-heimer’s disease.
4.4.2.1 Liposomes in Gene TherapyNearly, a quarter of all gene therapy trialsinvolve the use of plasmid DNA alone, which istypically very inefficient, or by complexingplasmid DNA with liposomes, which canimprove the rate of gene transfer to varyingdegrees. Liposomes are hollow microscopicspheres of phospholipid, and can be filled withDNA or other molecules during assembly. Theliposomes will merge with the membranes sur-rounding most animal cells and the contents ofthe liposome end up inside the cell, a processknown as Lipofection. Liposomes, both cationicand anionic, are currently being considered asvectors for gene therapy and their accomplish-ment in transfection is being studied by researchgroups and pharmaceutical companies(Fig. 4.5a). Lipofection is a promising approachbecause ‘‘armed’’ liposomes can be injected
directly into tumor tissue. In fact, liposomes areprobably of more use in delivering proteins thanDNA, something not feasible when using virusesas genetic engineering vectors. For example,toxic proteins such as tumor necrosis factor(TNF) can be packaged inside liposomes andinjected into tumor tissue (Fig. 4.5b). The lipo-somes merge with the cancer cell membranes,and the lethal proteins are then released insidethe cancer cells.
PEGylated liposome (PEGLip) technology isa new approach to improve the pharmacody-namic properties of therapeutic proteins. Insteadof encapsulating the drug, PEGLips are used ascarriers with the protein bound noncovalentlybut with high specificity to the outer surface.Unlike approaches such as mutagenesis, PEGy-lation, or fusion to carrier proteins, PEGLiptechnology does not involve changes to a pro-tein’s amino acid sequence and covalentattachment of stabilizing agents. PEGLip is anew platform technology that produces long-acting forms of coagulation factor VIII (FVIII)and activated FVII (FVIIa) and improves theefficacy of the hemophilia treatment (Suzukiet al. 2001).
Fig. 4.5 a Cationicliposomes (positivelycharged) are complexedwith DNA (plasmids); theliposome: DNA ratio isessential for optimaltransfection. b Anticanceragent delivered byLipofection. Liposomescan be filled with DNA/proteins such as TNF;interacts and merges withcell membrane and deliversinto the cell (http://www.azonano.com)
4 Gene Therapy 49
The development of liposomes as carriers fortherapeutic molecules is an ever-growingresearch area. The possibility of modulating thetechnological characteristics of the vesiclesmakes them highly versatile both as carriers ofseveral types of drugs (from conventional che-motherapeutics to proteins and peptides) and intherapeutic applications (from cancer therapy tovaccination).
4.4.2.2 Antisense TherapyAntisense RNA binds to the corresponding RNAand prevents its translation by ribosome. Genetherapy for certain disorders may result inoverproduction of some normal proteins. Hence,it is possible to treat these diseases by blockingtranscription using single-stranded nucleotidesequence that hybridizes with the specific geneand this is called antisense therapy. Antisensetherapy refers to the inhibition of translation byusing a single-stranded nucleotide and alsoinhibits both transcription and translation byblocking the transcriptional factor responsiblefor gene expression (Aartsma-Rus and vanOmmen 2007). Two main alternatives existwhen using antisense RNA.1. It is possible to use a full length anti-gene that is
transcribed to give a full length antisense RNA.The anti-gene must be carried on a suitablevector and expressed in the target cells.
2. Shorter artificial RNA oligonucleotides maybe used. An antisense RNA of 15–20 nucle-otides is capable of binding specifically topart of the complementary mRNA and pre-venting translation.Antisense oligonucleotides (oligos) can be
designed to complement a region of a particulargene or messenger RNA (mRNA) and serve aspotential blockers of transcription or translationthrough sequence-specific hybridization. Recentadvances in the use of oligodeoxynucleotide andplasmid-derived RNA as antisense agents are ofspecial relevance to cancer gene therapy whichhave lead to clinical trials and have proved
promising results. Transformed cell lines bearingplasmids and viruses designed for the transcrip-tion of antisense RNA have the advantage thatthey can be characterized thoroughly and theeffects of antisense RNA on target gene expres-sion and phenotype can be studied easily in vivo.The importance of examination of antisenseeffects in syngeneic and immunocompetent hostsis illustrated by the studies of insulin-like growthfactor and insulin-like growth factor receptorwhere tumor regression and protection againsttumor formation have been observed in cell types.
Antisense oligonucleotides are being investi-gated as a potential therapeutic modality that takesdirect advantage of molecular sequencing. Afundamental attraction of the antisense approachis that this method potentially may be applied toany gene product, in theory, for the treatment ofmalignant and nonmalignant diseases.
4.4.2.3 Ribozymes in Gene TherapyRecent advances of biological drugs havebroadened the scope of therapeutic targets andthis holds true for dozens of RNA-based thera-peutics currently under clinical investigation fordiseases ranging from genetic disorders to HIVinfection to various cancers.
RNA molecules that can serve as enzymes(catalytic ability) are referred to as ribozyme.Ribozymes have catalytic domains and recognizethe substrate RNA by base paring and cleaveRNA molecules. The naturally occurring ribo-zymes are to be modified, so that they can spe-cifically hybridize with mRNA sequences andblock protein synthesis. Thus, ribozymes mightbe engineered to recognize and destroy any targetmRNA molecule (Pelletier et al. 2006).
A vector carrying a gene encoding of theribozyme is used for delivery to the target cell.Transcription of this gene would result in pro-duction of the ribozyme RNA. The ribozymewould then bind and cleave the target mRNA.Genetically engineered ribozymes are a possibil-ity for treating certain cancer and viral diseases.
50 K. Rohini
4.4.2.4 RNA InterferenceRNA interference (RNAi) is a mechanism toselectively degrade mRNA expression hasemerged as a potential novel approach for drugtarget validation and the study of functionalgenomics. Like other biological drugs, RNAi-based therapeutics often requires a delivery vehicleto transport them to the targeted cells. Thus, theclinical advancement of numerous small interfer-ing RNAs (siRNA) drugs has relied on the devel-opment of siRNA carriers, includingbiodegradable nanoparticles, lipids, bacteria, andattenuated viruses. Most therapies permit systemicdelivery of the siRNA drug, while others useex vivo delivery by autologous cell therapy. Themost widely used siRNA delivery methodologyconsists of cationic lipids or cationic polymers thatpackage siRNA into stable nanoparticles capableof translocation across the cellular membrane.Studies have shown that this targeted nanoparticleformulation with improved tissue specificity anddelivery efficiency silenced the target gene in themetastasis effectively without any significantimmunotoxicity and hence proved to be a potentialtool for clinical cancer therapy.
4.5 Landmarks in Gene Therapyfor Genetic Diseases
Clinical trials are becoming more increasinglyprevalent. The most interesting is that gene ther-apy seems to have cured 8 of 10 children who hadpotentially fatal ‘‘bubble boy disease’’. Bubbleboy disease is formally called severe combinedimmunodeficiency, or SCID, a genetic disorderdiagnosed in about more than 40 babies each yearin the United States. The name ‘‘bubble boy’’ isgiven after a Houston boy, David Vetter who wasborn with a genetic disorder leaving him no nat-ural immunities against disease, became famousfor living behind plastic barriers to protect himfrom germs. He died at the age of 12 in 1984.
Selected list of genetic diseases that are likelyto be cured by gene therapy including SCID isSickle cell anemia, Fanconi anemia, Thalassemia,
Gausher’s disease, Hunter disease, Hurler dis-ease, Osteoporosis and Chronic granulomatousdisease.
4.5.1 Therapy for AdenosineDeaminase Deficiency
The first human gene therapy was carried out tocorrect the deficiency of the enzyme ADA onSeptember 14, 1990 at the National Institute ofHealth. Severe combined immunodeficiency(SCID) is a rare inherited immune disorder asso-ciated with T-lymphocytes and to a lesser extent B-lymphocytes dysfunction. According to Aiuti et al.(2009) in the deficiency of ADA, deoxyadenosineand its metabolites accumulated and destroyed T-lymphocytes. Both the lymphocytes participate inthe defence mechanism of body, thus the patient ofSCID (lacking ADA) suffers from infectious dis-eases. The technique for the treatment for ADAdeficiency is as follows:• A plasmid vector bearing proviral DNA is
selected.• A part of proviral DNA is replaced by the
ADA gene and the gene (G 418) coding forantibiotic resistance and then cloned.
• Circulating lymphocytes are removed fromthe patient suffering from ADA deficiency.
• These cells are transfected with ADA gene byexposing to retrovirus carrying the said gene.
• The genetically modified lymphocytes aregrown in cultures to confirm the expression ofADA gene and returned to the patient.These lymphocytes persist in the circulation
and synthesize ADA. Thus, the ability of thepatient to produce antibodies is increased. Themain drawback is that the lymphocytes have ashort life span; hence the transfusions have to becarried out frequently.
4.5.2 Therapy for Lesch–NyhanSyndrome
Lesch–Nyhan is an inborn error in purinemetabolism due to the defect in a gene that
4 Gene Therapy 51
encodes for the enzyme hypoxanthine-guaninephosphoribosyl transferase (HGPRT). In theabsence of HGPRT, purine metabolism is dis-turbed and uric acid levels increases resulting insevere gout and kidney damage. Use of retroviralvectors, the HGPRT producing genes is success-fully inserted into the bone marrow for treatment.
4.5.3 Therapy for FamilialHypercholesterolemia
The patients with familial hypercholesterolemialack the low density lipoprotein (LDL) receptorson their liver cells. As a result, the LDL cho-lesterol is not metabolised in liver, leading toarterial blockage and heart diseases. The hepa-tocytes were transduced with retroviruses car-rying genes for LDL receptors, which areinfused into patient’s liver. The hepatocytesestablish and produce functional LDL receptors.
4.5.4 Therapy for Cystic Fibrosis
CF is a fatal genetic disease, characterized by theaccumulation of sticky dehydrated mucus in therespiratory tract and lungs. Due to this, thepatients of CF are highly susceptible to bacterialinfections in their lungs. In normal persons, thechloride ions of the cells are pushed out throughthe participation of a protein called cystic fibrosistransmembrane regulator (CFTR). In patients ofCF, the CFTR protein is not produced due to thegene defect. As a result, the respiratory tract andthe lungs become dehydrated with sticky mucus,which serves as an ideal environment for bacte-rial infections. Adeno-associated virus vectorsystem showed some encouraging results in thegene therapy for CF (Conese et al. 2011).
4.5.5 Therapy for Cancer
Cancer is a leading cause of death throughoutthe world and gene therapy is the latest andnovel approach for cancer treatment.
4.5.5.1 Tumor Necrosis FactorIt is a protein produced by human macrophageswhich provide defense against cancer cells. Aspecial type of immune cells, the tumor infil-trating lymphocytes (TILs), enhance the cancerfighting ability. These were transformed with theTNF gene (along with neomycin resistant gene)and used for the treatment for malignant mela-noma (Woods and Bottero 2006).
4.5.5.2 Suicide Gene TherapySuicide gene therapy begins by delivering atherapeutic gene into the cancer cells. The geneencodes an enzyme that converts nontoxic pro-drug into a toxic compound. The gene encodingthe enzyme thymidine kinase (TK) is often refer-red to as suicide gene and is used for the treatmentfor certain cancers. TK phosphorylates nucleo-sides which are used for synthesis of DNA duringcell division. The drug ganciclovir (GCV) bears aclose structural resemblance to certain nucleo-sides (thymidine). The nontoxic prodrug, thenucleoside analog ganciclovir, is converted intoits monophosphate by thymidine kinase, becauseonly the cancer cells have thymidine kinase andall the noncancerous cells are unaffected. Normalcellular enzymes then convert the monophosphateto ganciclovir triphosphate (GCV-TP). This actsas a DNA chain terminator. DNA polymeraseincorporates GCV-TP into the growing strands ofDNA and kills the cancer cells (bystander effect).In suicide gene therapy, the vector used is theherpes simplex virus (HSV) with the gene forthymidine kinase inserted in its genome. Normalbrain cells do not divide while the brain tumorcells undergo unchecked division. Studies byDeanna and Burmester (2006) have proved that byusing GCV-HSVTK suicide gene therapy theproliferation of the cells can be reduced.
4.5.5.3 Gene Replacement TherapyA gene named p53 codes for a protein withmolecular weight of 53 kilo Daltons, hence thename p53. p53 is considered to be a tumorsuppressor gene, since the protein it encodes
52 K. Rohini
binds with the DNA and inhibits replication. Thetumor cells of several tissues were found to havealtered genes of p53, and a main causative factorin tumor development. Researchers have foundencouraging results by employing adenovirusvector system to replace the damaged p53 by anormal gene.
4.5.6 Therapy for AIDS
AIDS is a global disease and invariably a fatalone with no cure. Researches have madeattempts to relieve its effects through genetherapy which are discussed below:
4.5.6.1 rev and env GenesA mutant strain of HIV lacking rev and envgenes has been developed. Due to the lack ofthese genes, the virus cannot replicate, henceused for therapeutic purposes. T-lymphocytesfrom HIV-infected patients are removed andmutant viruses are inserted into them. Themodified T-lymphocytes are cultivated andinjected into the patients. Due to the lack ofthe genes the viruses cannot multiply, but theycan stimulate CD8 killer lymphocytes, whichare proved to destroy HIV-infected cells(Cavazzana-Calvo et al. 2000).
4.5.6.2 Genes to Activate gp120gp120 is a glycoprotein with molecular weightof 120 kilo Daltons, which is present in theenvelope of HIV. It is essential for binding ofvirus to the host for replication. Researches havesynthesized a gene F105 to produce an antibodythat can inactivate gp120. In the anti AIDStherapy, HIV cells were engineered to produceanti-HIV antibodies (Patterson et al. 2009).
4.6 Ethical Issues
One of the main concerns regarding gene ther-apy was that genetic material used to treatsomatic cells would find its way into sperm andova, thus affecting offspring of the patient.
However, careful selection of vectors and targetcells prevents the spread of genetic material togerm cells. Trials to define limits of safety andefficacy in somatic cell gene therapy have beenunderway for the better part of a decade. Beyondsafety and efficacy issues, ethical concerns insomatic gene therapy research are familiar inmany clinical settings, weighing potential harmsand benefits, selection of patients for research,assurance that consents to experimental treat-ments is informed and voluntary, and protectionof privacy and confidentiality of medical infor-mation. Ethical concerns about germ line ther-apy have been widely discussed, especiallystarting with the beginning of the Human Gen-ome Project. Germ line gene therapy is far moretechnically difficult than somatic cell therapy.These concerns have raised queries about whyresearch in germ line therapy should be pursuedat all.
4.7 Conclusion
Gene therapy is moving on to higher gear for thetreatment for a number of genetic diseases.
Gene therapy studies have shown promisingresults in the treatment of HIV and cancer.Ethical stem cells may be helpful to treatpatients with osteogenesis; Red Orbit genetherapy may improve some form of blindnessand successful clinical trials for Parkinson’sdisease are few examples of gene therapy suc-cess. Besides, researchers have engineeredpatients’ own pathogen fighting T-cells to targeta molecule on the surface of leukemia cells. Thealtered T-cells were grown outside the body andinfused back to the patient suffering from late-stage chronic lymphocytic leukemia (CLL).
Theoretically, gene therapy is the permanentsolution for genetic disease. It broadly involvesisolation of a specific gene, making its copies,inserting them into target tissue cells to make thedesired protein. The most significant factor is toensure the gene is harmless to the patient and isexpressed appropriately. Another importantconcern is the reaction of body’s immune systemto the foreign proteins.
4 Gene Therapy 53
It is true that gene therapy has several limi-tations, despite intensive research. Butresearchers are persistently working and waitingfor a great breakthrough to revolutionize theworld with the new medicine.
References
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Aiuti A, Cattaneo F, Galimberti S, Benninghoff U,Cassani B, Callegaro L, Scaramuzza S, Andolfi G,Mirolo M, Brigida I (2009) Gene therapy for immu-nodeficiency due to adenosine deaminase deficiency.N Engl J Med 360(5):447–458
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Bushman FD (2007) Retroviral integration and humangene therapy. J Clin Invest 117(8):2083–2086
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G,Gross F, Yvon E, Nusbaum P, Selz F, Hue C, CertainS, Casanova JL (2000) Gene therapy of human severecombined immunodeficiency (SCID)-X1 disease.Science 288(5466):669–672
Conese M, Ascenzioni F, Boyd AC (2011) Gene and celltherapy for cystic fibrosis: from bench to bedside.J Cystic Fibr 10(2):S114–S128
Deanna C, Burmester JK (2006) Gene therapy for cancertreatment: past, present and future. Clin Med Res4(3):218–227
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Mandel RJ, Manfredsson FP, Foust KD, Rising A,Reimsnider S, Nash K, Burger C (2006) Recombinantadeno-associated viral vectors as therapeutic agents totreat neurological disorders. Mol Ther 13:463–483
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Pelletier R, Caron SO, Puymirat J (2006) RNA basedgene therapy for dominantly inherited diseases. CurrGene Ther 6:131–146
Patterson S, Papagatsias T, Benlahrech A (2009) Use ofadenovirus in vaccines for HIV. Handb Exp Pharma-col 275–293
St George JA (2003) Gene therapy progress and pros-pects: adenoviral vectors. Gene Ther 10:1135–1141
Suzuki S, Tadakuma T, Kunitomi M, Takayama E, SatoM, Asano T (2001) Liposome-mediated gene therapyusing HSV-TK/Ganciclovir under the control ofhuman PSA promoter in prostate cancer cells. UrolInt 67:216–223
Weidhaas JB, Angelichio EL, Fenner S, Coffin JM (2000)Relationship between retroviral DNA integration andgene expression. J Virol 74(18):8382–8389
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54 K. Rohini
5RNA Interference and Its Applications
Jyoti Saxena
Abstract
RNA interference (RNAi) refers to a group of post-transcriptional ortranscriptional gene silencing mechanisms conserved from fungi tomammals. It is a phenomenon primarily for the regulation of geneexpression, self or nonself depending upon the surrounding factors orconditions. It is done in nature with the help of noncoding RNAmolecules to control cellular metabolism and helps in maintaininggenomic integrity by preventing the invasion of viruses and mobilegenetic elements. It is a simple and rapid method of silencing geneexpression in a range of organisms as a consequence of degradation ofRNA into short RNAs that activate ribonucleases to target homologousmRNA. The process of RNAi can be mediated by either small interferingRNA (siRNA) or micro RNA (miRNA). The RNAi pathway is triggeredby the presence of double-stranded RNA, which is cleaved by theribonuclease-III domain-containing enzyme Dicer to generate 20–25nucleotide long siRNA duplexes. siRNA is then loaded onto the RNA-induced silencing complex (RISC), in which an Argonaute (Ago)-familyprotein, guided by the siRNA, mediates the cleavage of homologousRNAs. Synthetic double-stranded RNA (dsRNA) introduced into cellscan selectively and robustly induce suppression of specific genes ofinterest. Because of its exquisite specificity and efficiency, RNAi is beingconsidered as valuable research tool, not only for functional genomics,but also for gene-specific therapeutic activities that target the mRNAs ofdisease-related genes.
5.1 Introduction
Gene silencing is a general term that describesepigenetic processes of gene regulation. It refersto ‘switching off’ of a gene by a mechanism otherthan genetic mutation. That is, a gene whichwould be expressed under normal circumstances
J. Saxena (&)Biochemical Engineering Department, BT KumaonInstitute of Technology, Dwarahat, Uttarakhand263653, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_5, � Springer India 2014
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is switched off by machinery in the cell. Genesilencing may be a part of an ancient immunesystem protecting the genome of the organismsfrom infectious DNA elements such as transpo-sons and viruses. RNA interference, commonlyknown as RNAi, regulates gene silencing mostlyeither at the transcriptional or post-transcrip-tional level. It is a sort of ‘‘Reverse Genetics.’’
RNAi is the mechanism in molecular biologywhere the presence of certain fragments ofdouble-stranded RNA (dsRNA) interferes withthe expression of a particular gene which sharesa homologous sequence with the dsRNA. Inother words, it is a process of using specificsequence of dsRNA to knock down the expres-sion of complementary gene. This phenomenonis distinct from other gene silencing methods inthat silencing can spread from cell to cell andgenerate heritable phenotypes in first generation.It only functions in eukaryotes as prokaryoticRNase III degrades shorter fragments of only 12base pairs. RNAi is now known to operate inhumans, mice and other mammals, as well as infrogs, fruit fly, hydra, trypanosomes, zebra fish,fungi, and plants.
In early 1990s horticultural researcher Rich-ard Jorgensen and his team mates experimentedon petunias by introducing numerous copies of agene that encoded deep purple pigmentation inflowers. But to their surprise unexpected oppo-site result was achieved in the form of white orpatchy flowers in place of purple colouration(Napoli et al. 1990). They concluded thatsomehow the introduced gene had silencedthemselves as well as plants’ endogenous purplepigment gene. They called this as co-suppressionof gene expression but the molecular mechanismremained unknown.
A few years later, similar results of genesilencing were obtained in the experiments whengenetically engineered RNA viruses containingplant gene were introduced into plants. Both thetransgene and normal cellular copy of geneswere turned off. This phenomenon was calledVIGS, i.e., ‘‘virus induced gene silencing.’’ Atthat time, these observations were puzzling untilin 1998 Andrew Fire and Craig Mello at theCarnegie Institution of Washington and the
University of Massachusetts Cancer Centre,respectively first described RNAi phenomenonin round worm, Caenorhabditis elegans byinjecting dsRNA. They also coined the termRNAi (Fire et al. 1998). For their revolutionarywork on RNAi they shared the Nobel Prize inmedicine in 2006.
There are two classes of RNA moleculescalled small (short) interfering RNA (siRNA)and micro RNA (miRNA). siRNA can bedefined as a class of dsRNAs consisting ofapproximately 18–23 nucleotides in length,generated from long dsRNAs by the action of anuclease enzyme called Dicer. Its antisensestrand functions in gene silencing by promotingthe cleavage of mRNA. miRNA, on the otherhand is a class of 19–25 nucleotides, single-stranded RNA encoded in genomes of most ofthe multicellular organisms. They vary from afew thousand to 40,000 molecules per cell. Theyare encoded by the genome but unlike mRNA,do not translate a protein, instead work for theregulation of the expression of mRNAs. LikesiRNA, these are also produced by the cleavageof dsRNAs by Dicer.
RNAi has become a valuable research tool,both in in vitro studies and living organismsbecause synthetic dsRNA introduced into cellscan selectively and robustly induce suppressionof specific genes of interest. RNAi may be usedfor large-scale screens that systematically shutdown each gene in the cell, which can helpidentify the components necessary for a partic-ular cellular process or an event such as celldivision. Besides, it has various applications inmedicine, biotechnology, and functionalgenomics.
5.2 Small Interfering RNA
siRNAs are short double-stranded RNA mole-cules that induce sequence specific post-tran-scriptional gene silencing (PTGS). Their rolewas first discovered in 1999 by David Baul-combe’s group in plants. siRNAs have a well-defined structure: two hybridized strands of18–23 RNA bases (usually 21 bp), each with
56 J. Saxena
phosphorylated 50 ends and hydroxylated 30 endswith two overhanging DNA nucleotides at the 30
terminus. The easiest way to achieve RNAi isthe use of synthetic siRNA molecules. OncesiRNA is transfected to a cell, it is incorporatedinto a nuclease complex called RNA-inducedsilencing complex (RISC). RISC targets andcleaves mRNA that is complementary to thissiRNA, thus interrupting translation of targetedgenes. The sequence specificity of siRNA mol-ecules allows specific silencing of about 90 % ofknown genes.
siRNA plays many roles, but most notable isin the RNAi pathway, where it interferes withthe expression of specific genes with comple-mentary nucleotide sequence. It offers an effi-cient and easy method for post-transcriptionalsequence specific silencing of genes and pre-sents a powerful new tool to study gene function,target validation, and signal transduction.Besides, its applications are also in drug dis-covery and gene therapy.
There are many challenges encountered insiRNA research. Occasionally it has been foundthat nonspecific effects are triggered by theexperimental introduction of a siRNA. When amammalian cell comes across a double-strandedsiRNA, it is mistaken for a viral by-product andmounts an immune response.
5.3 Micro RNA
miRNAs are noncoding genes present in cells ofvarious eukaryotic organisms. These are a formof single-stranded RNA, typically 19–25 nucle-otides long, and are thought to regulate theexpression of other genes. These are RNA geneswhich are transcribed from DNA but do notform a protein. They were discovered in 1993 byLee and Feinbaum during a study of the genelin-14 in C. elegans development. The DNAsequence that codes for miRNA is sometimesmuch longer than miRNA itself and includes notonly the miRNA sequence but also an approxi-mate reverse complement. Processing alsoinvolves components of the RNAi machinery.Once processed they seem to bind to their target
mRNAs, however they are not 100 % comple-mentary, a couple of base mismatches may beseen. The primary transcript known as apri-miRNA is processed in the cell nucleus andforms a 70-nucleotide stem-loop structure calleda pre-miRNA. This dsRNA hairpin loop or stemloop is formed due to pairing of single-strandedmiRNA sequence and its reverse complementbases. In animals, RNAs III named as Drosha isused to cut the base of hairpin loop to release thepre-miRNA. This pre-miRNA is then trans-ported into the cytoplasm by a carrier protein,Exportin 5. 20–25 nucleotides are removed byDicer to release the mature miRNA. However, inplants (since Drosha has not been reported) theprocessing takes place through Dicer in nucleus.
The function of miRNAs appears to be ingene regulation. For that purpose, a miRNA iscomplementary to a part of one or moremRNAs. Gene silencing may occur either viamRNA degradation or by preventing mRNAfrom being translated. Since its discovery,miRNA research has revealed its multiple rolesin negative regulation (transcript degradationand sequestering, translational suppression) andpossible involvement in positive regulation(transcriptional and translational activation). Byaffecting gene regulation, miRNAs are likely tobe involved in most biological processes. miR-NAs occasionally also cause histone modifica-tion and DNA methylation of promoter sites,which affects the expression of target genes. Thehuman genome may encode over 1,000 miR-NAs, which may target about 60 % of mam-malian genes.
The process of RNAi can be moderated byeither siRNA or miRNA but there are subtledifferences between the two. siRNA is consid-ered exogenous double-stranded RNA that istaken up by cells or enters via vectors likeviruses, while miRNA is single stranded andcomes from endogenous noncoding RNA foundwithin the introns of larger RNA molecules.Mature miRNAs are structurally similar to siR-NAs, however they undergo extensive posttranscriptional modifications before reachingmaturity. Thus, miRNA and siRNA share thesame cellular machinery downstream of their
5 RNA Interference and its Applications 57
initial processing. The siRNAs derived fromlong dsRNA precursors also differ from miR-NAs in that miRNAs, especially those in ani-mals, typically have incomplete base pairing to atarget and inhibit the translation of many dif-ferent mRNAs with similar sequences. In plants,however, miRNA tends to have a more perfectlycomplimentary sequence which induces mRNAcleavage as opposed to just repression of trans-lation. In contrast, siRNAs typically base-pairperfectly and induce mRNA cleavage only in asingle, specific target. Both siRNA and miRNAcan play a role in epigenetics through a processcalled RNA-induced transcriptional silencing(RITS). Likewise, both are important targets fortherapeutic use because of the roles they play incontrolling the gene expression.
5.4 Components of Gene Silencing
Many important enzymes and genes encodingthem have been identified in various modelorganisms which have been identified by anal-ysis of mutants. Some of the components iden-tified serve as initiators, while others aseffectors, amplifiers, and transmitters of the genesilencing process. These are described asfollows.
5.4.1 RNase III
RNase III is divided into three classes on thebasis of domain structure viz. bacterial RNaseIII, Drosha family nucleases, and Dicer enzyme.Dicer was identified in Drosophila extract whichshowed its involvement in the initiation ofRNAi. It was named as Dicer (DCR) due to itsability to digest dsRNA into uniformly sizedsmall RNAs (siRNA) by Hannon and a graduatestudent in his laboratory, Emily Bernstein in2000 (Bernstein et al. 2001). In Drosophila,Dicer which is a large multidomain RNase IIIenzyme has been identified in existence into twoforms: Dcr-1/Loquacious (Loqs)-PB (alsoknown as R3D1-L[long]) that creates miRNA
and Dcr-2/R2D2 that generates siRNA. Dcr-1shares a structural homology with Dcr-2, despitethat it displays different sets of properties suchas ATP requirements and substrate specifica-tions. Dcr-1 is an enzyme that shows ATP-independent functions and affinity toward stem-loop form of RNA and requires a dsRNA bind-ing protein partner. Dcr-2 on the other handshows ATP-dependent activity with substratespecificity to dsRNA. It also requires a dsRNAbinding protein, namely R2D2, which functionin association with specific RNase enzyme Dcr-2 forming a heterodimeric complex.
Dicer has four distinct domains such as anamino-terminal helicase domain, dual RNase IIImotifs, a dsRNA binding domain, and a PAZdomain. Cleavage by Dicer is thought to becatalyzed by its tandem RNase III domain.Complete digestion by RNase III enzyme resultsin dsRNA fragments of 12–15 bp, half the sizeof siRNAs. The RNase III enzyme acts as adimmer, thus digests dsRNA with the help oftwo compound catalytic centers, whereas eachmonomer of the Dicer enzyme possesses twocatalytic domains, with one of them deviatingfrom the consensus catalytic sequences(Agarwal et al. 2003).
5.4.2 RNA-Induced Silencing Complexand Guide RNAs
The RISC is a multiprotein complex that incor-porates one strand of a siRNA or miRNA. Afterrecognizing the complementary mRNA, it acti-vates RNase to cleave the RNA. The inability ofcellular extracts treated with a Ca2+-dependentnuclease (micrococcal nuclease, which candegrade both DNA and RNA) to degrade thecognate mRNAs and the absence of this effectwith DNase I treatment showed that RNA wasan essential component of the nuclease activity.Hammond et al. (2001) termed the sequence-specific nuclease activity observed in the cellularextracts responsible for ablating target mRNAsas RISC. The RNA endonuclease, Dicer plays arole in aiding RISC action by providing the
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initial RNA material to activate the complex aswell as the first RNA substrate molecule. Whenthe Dicer, which has endonuclease activityagainst dsRNAs and pre-miRNAs, cleaves a pre-miRNA stem-loop or a dsRNA, a 19–25-basepair dsRNA fragment is formed with a two-nucleotide 30 overhang at each end.
Small RNAs are found to be associated withsequence-specific nuclease, which serves asguides to target-specific messages based uponsequence recognition. The multicomponentRNAi nuclease has been purified to homogeneityas a ribonucleoprotein complex of &500 kDa.One of the protein components of this complexbelonged to Argonaute family of proteins andcalled as Argonaute2 (AGO2). AGO2 is homol-ogous to RDE1, a protein required for dsRNA-mediated gene silencing in C. elegans. AGO2 isa &130 kDa protein containing polyglutamineresidues, PAZ and PIWI domains characteristicof members of the Argonaute gene family. TheArgonaute family members have been linkedboth to the gene-silencing phenomenon and tothe control of development in diverse species.
5.4.3 RNA-Dependent RNAPolymerase
The effects of RNAi are potent and systemic innature. This has led to a proposed mechanism inwhich RNA-dependent RNA polymerases(RdRPs) play a role in both triggering andamplifying the silencing effect. An excessamount of dsRNA is considered better for theformation of more 21–23 nt dsRNAs to survivedilution by cell division. This dsRNA signal isamplified in nature by an RdRP activity. RdRPprobably converts an aberrant ssRNA populationinto dsRNA or repeatedly copy the dsRNAs soas to produce a population of ssRNAs that couldthen interact with target RNA.
In plants, the DNA-dependent RNA poly-merase IV (Pol IV) is important for siRNAproduction and RNAi-directed transcriptionalsilencing. However, Pol IV homologues are not
found in fungal or animal genomes. In yeast,mutants of the RNA polymerase II (Pol II)subunits show loss of centromeric siRNA andRNAi-dependent heterochromatin formation atthe centromeric repeat regions, probably bycoupling transcription with transcriptionalsilencing machinery. It is not known whether PolII plays a similar role in post-transcriptionalsilencing and in the silencing of other chromo-somal regions.
5.4.4 RNA and DNA Helicases
A diverse array of proteins specific for most ofthe eukaryotic organisms seems to carry outelimination of aberrant RNA. Broadly, they fallin the biochemically similar group of RNA-DNAhelicases. A strain of C. reinhardtii carrying mut-6 mutation was found to relieve silencing by atransgene, and by also activating the transposons.The deduced MUT6 protein contains1,431 amino acids and is a member of the DEAHbox RNA helicase family. In Neurospora crassa,three classes of quelling-defective mutants(qde1, qde2, and qde3) have been isolated. Theqde3 gene has been cloned, and the sequenceencodes a 1,955 amino-acid protein. The proteinshows homology with several polypeptidesbelonging to the family of RecQ DNA helicases,which includes the human proteins for Bloom’ssyndrome and Werner’s syndrome. In addition,QDE3 protein is believed to be involved in theactivation step of gene silencing.
Seven smg genes involved in nonsense-med-iated decay have been identified from themutants of C. elegans. The SMG proteins couldunwind dsRNA to provide a template foramplification activity. In this way, the three SMGproteins might facilitate amplification of thesilencing signal and cause persistence of thesilenced state. Alternatively, SMG proteins couldincrease the number of dsRNA molecules bypromoting endonucleolytic cleavage of existingdsRNA molecules, which has been observed inDrosophila flies. No SMG2 homologues have
5 RNA Interference and its Applications 59
been identified in plants or fungi. However, asearch of the Arabidopsis thaliana genomesequence database revealed a number of candi-dates with either helicase and/or RNase domains.
5.4.5 Trans-Membrane Protein(Channel or Receptor)
It is well established now that in a number oforganisms the spread of gene silencing from onetissue to another is systemic in nature. A greenfluorescent protein (GFP) was identified from aspecial transgenic strain of C. elegans HC57.Out of 106 sid mutants belonging to threecomplementation groups (sid1, sid2, and sid3),sid1 mutant was characterized which failed toshow systemic RNAi. The SID1 polypeptide ispredicted to be a 776 amino-acid membraneprotein consisting of a signal peptide and 11putative trans-membrane domains. Based on thestructure of SID1, it was suggested that it mightact as a channel for the import or export of asystemic RNAi signal or might be necessary forendocytosis of the systemic RNAi signal, per-haps functioning as a receptor.
5.5 Mechanism
Our knowledge on RNAi has expanded dra-matically in short time since its discovery. In thelast few years, important insights have beengained in elucidating the mechanism of RNAi,although it seems to be very complicated. RNAiappears to be a highly potent and specific pro-cess, which starts when a dsRNA is introducedeither naturally or artificially in a cell. An en-doribonuclease enzyme cleaves the long dsRNAinto small pieces of miRNA or siRNA depend-ing upon the origin of long dsRNA, i.e.,endogenous or exogenous, respectively. AdsRNA may be generated by either RNA-dependent RNA polymerase or bidirectionaltranscription of transposable elements.
Based on the in vitro and in vivo experimentalresults, a two-step mechanistic model for RNAihas been established (Fig. 5.1). The first step,
referred to as the RNAi initiating step, involvesthe binding of RNA nucleases to a large dsRNAand its cleavage into discrete &18- to &23-nucleotide RNA fragments (siRNA). In thesecond step, these siRNAs join a multinucleasecomplex, RISC, which degrades the homologoussingle-stranded mRNAs.
5.5.1 Initiation
This stage is characterized by generation ofsiRNA mediated by type III endonucleaseDicer. It appears that the RNAi machinery,once it finds RNA duplexes, cuts it up intosmall molecules with a length of about 21 nt.These also have 2-nucleotide, 30 overhangs,and 50 phosphates. Of course, different organ-isms have different numbers of Dicer genesthat process different sorts of dsRNAs. InDrosophila, Dicer-1 is seen to interact with thedsRBD protein Loquacious (Loqs). Immuno-affinity purification experiments have revealedthat Loqs resides in a functional pre-miRNAprocessing complex, and stimulates and directsspecific pre-miRNA processing activity. Dicer-2 requires R2D2, a dsRNA binding protein,which unlike Loq is supposed to be composedof two dsRNA binding domains. Both dsRNAbinding domains of Dcr-2 and R2D2 are crit-ical for Dcr-2/R2D2 complex to bind and loadsiRNA into siRISC complex.
In general, Dicer works by recognizing theends of dsRNA with its PAZ domain (Dicer hasPAZ domain at one end and dual RNase IIIdomains on the opposing end) and then cuttingthe dsRNA with its RNase III domain. Thedistance between the PAZ and RNase III domaindetermines the length of siRNA produced byDicer which varies by species. Long dsRNA andmiRNA precursors are processed to siRNA/miRNA duplexes by the RNase-III-like enzymeDicer. These short dsRNAs are subsequentlyunwound and assembled into effector com-plexes, RISCs, which can direct RNA cleavage,mediate translational repression or induce chro-matin modification.
60 J. Saxena
5.5.2 Effector
The second step of RNAi mechanism is referredas effector step; the incorporation of guidestrand. This is characterized by the formation ofsilencing complex. The siRNA and miRNAduplexes that contain ribonucleoprotein particles
(RNPs) are now made into RISC. Generally,effector complexes containing siRNAs areknown as a RISC, while those containing miR-NAs are known as miRNPs.
It is thought that RISC undergoes an ATP-dependent step that activates the unwinding ofthe double-stranded siRNAs. RISC is composedof PPD proteins (PAZ PIWI Domain proteins),which are highly conserved super-family.Members of PPD proteins contain PAZ (100amino acids) and C-Terminal located PIWI (300amino acids). The Argonaute protein and onestrand of the siRNA form the RISC. Once theRISC complex has been formed from 50-phos-phorylated siRNAs and endogenous Argonauteprotein, the siRNAs in the RISC complex guidethe degradation that is sequence-specific, of thecomplementary or near complementary mRNAs.RISC binds to the mRNA which is targeted bythe single RNA strand within the complex andcleaves the mRNA or represses their translationby homology-dependent m-RNA degradation.mRNA is cleaved in the middle of its comple-mentary region. This cleaved mRNA cannot betranslated. These newly synthesized siRNAsdestroy the target with the help of RNA-dependentRNA polymerase (RdRP). Afterwards RISCdissociates and is ready to cleave other mRNAs.By that even a few numbers of the RISC canlead to high-level gene silencing. The siRNAderived in RNAi pathway may have two differ-ent fates, either it may be converted into holo-RISC for the destruction of the target m-RNA, orit may contribute to the amplification step by thegeneration of secondary siRNA.
RISC and miRNP complexes work by cata-lyzing hydrolysis of the phosphodiester linkageof the target RNA. The mechanism by whichrepression of translation guided by miRNAworks, is not as well understood. It has beensuggested that miRNAs affect translation ter-mination or elongation rather than actual initia-tion of the process. Besides, miRNAs can act assiRNAs and vice versa. Perhaps mRNA degra-dation and translational regulation guided bymiRNAs are used as simultaneous mechanismsfor natural regulation.
Fig. 5.1 Mechanism of RNA interference
5 RNA Interference and its Applications 61
5.6 Delivery Methods
RNAi has rapidly advanced from a laboratoryobservation into a major area of research withinbiology and medicine. The RNAi treatments areentering various stages of clinical trials. How-ever, development of RNAi-based agents hasbeen hindered because siRNAs are unstable inserum and delivery across the cell membrane ishighly inefficient. Numerous methods have beendeveloped to facilitate delivery of RNAi inanimals and patients, each with their own set ofadvantages and disadvantages. To improve theeffect of RNAi-based therapy, the enzymaticstability and cellular uptake of siRNA should besignificantly enhanced, while their immunoacti-vation should be decreased. The two mostcommon approaches for RNAi delivery are lipidmediated transfection and viral mediated trans-duction. Typically, researchers strive to achievethe highest levels of transfection efficiencypossible. Transfection efficiency describes thepercentage of cells that have received the RNAiduplex or expression plasmid. For many diseasemodels, the most desirable cell types to use areprimary cultures. However, these cannot betransfected adequately with commerciallyavailable transfection reagents. A powerfulalternative to cationic lipid-mediated transfec-tion is viral delivery of vectors expressing RNAisequences. This option is the best for delivery toprimary, hard-to-transfect, and non-dividingcells. Viral delivery can also be used to createstable cell lines with inducible RNAi expressionor to express RNAi sequences with tissue-spe-cific promoters.
5.6.1 Viral Methods
Since viruses work by delivering genetic mate-rial into a cell, scientists have been viewingthem as a potential method of delivering DNA orRNA into a cell since long. Lentiviruses, afamily of retroviruses that includes the humanimmunodeficiency virus (HIV), use RNA astheir genetic material and have been used to
deliver RNA to the cells of live mammals(including humans) for RNAi. Lentiviruses arevalued among retroviruses for this purposebecause they are the only ones that can work innon-dividing cells. Other viruses may also beused for RNAi delivery. Adenoviruses and her-pes simplex-1 virus, both of which use double-stranded DNA instead of RNA, have beenstudied as RNAi vectors. Jonge et al. (2006)utilized virosomes derived from influenza as ameans of delivering siRNA in vivo. Virosomes,which are empty influenza virus envelopes werecomplexed with cationic lipids for protectionfrom nuclease activity and, following intraperi-toneal injection, were demonstrated to success-fully encapsulate siRNA duplexes and deliverthem to cells in peritoneal cavity of mice.Virosomes may also serve as an effective meansto target cells of the respiratory epithelium andcells that possess Fc receptors, such as dendritic-cells, macrophages, and natural-killer cells.Viral vectors have also been used to suppress theexpression of HIV human co-receptor, chemo-kine receptor 5 to prevent HIV infection topenetrate lymphocytes. Such technologies areundergoing tests in a clinical trial (Nguyen et al.2008).
Despite initial promise as a vehicle for thedelivery of RNAi into live animals, viral vectorsno longer appear to be the tool of choice due toadverse effects observed in viral vector genetherapy trials, and RNAi-induced hepatotoxicity.
5.6.2 Liposomal Delivery
For many immortalized cell lines, transfectionwith a lipid- or amine-based reagents is thepreferred option. A major focus in the develop-ment of RNAi therapeutics is liposomal formu-lations for siRNA delivery to improve efficacyand safety. Many commercial institutions asreported by Nguyen et al. (2008) such as SilenceTherapeutics plc are in the process of improvingliposomal delivery. The work is in progress on anovel class of lipid delivery vehicles using ‘A-tuPLEX,’ a mixture of cationic and fusogenic
62 J. Saxena
lipids complexed with negatively charged siR-NAs, to promote uptake of siRNAs into endo-thelial cells of blood vessels in the liver andtumors.
Protiva Biotherapeutics Inc and Alnylamhave utilized stable nucleic acid lipid particle(SNALP)-formulated siRNAs. SNALPs arespecialized lipid nanoparticles that encapsulatesiRNAs and are coated with a diffusible PEG-lipid conjugate which stabilizes the particleduring formulation by providing a neutralhydrophilic exterior and prevents rapid systemicclearance in vivo by shielding the cationicbilayer; therefore, SNALPs facilitate the cellularuptake and endosomal release of the particle’ssiRNA. In addition, Alnylam is exploring alibrary of ‘lipidoids’ or lipid-like moleculesdeveloped by Robert Langer and DanielAnderson, to develop siRNA therapeutics fortwo different disease targets in the liver: hyper-cholesterolemia and liver cancer. Chemicalmodification and bioconjugation with lipids areknown to improve the stability and cellularuptake of siRNA.
5.6.3 Chemical Modification
Chemical modifications to sugars, backbones, orbases of siRNAs have been shown to enhancetheir stability, prevent them from triggering animmune response, control their pharmacokineticprofiles and reduce nonspecific effects withoutaffecting their biological activity. Most com-monly used chemical modifications of siRNAinclude phosphorothiolation (P = S) of the non-bridging oxygen at the 30-end, 20-sugar modifi-cation (such as 20-OMe or 20-F) and lockednucleic acid (LNA). Chemical modifications ofsiRNA confer enhanced gene silencing at lowerdoses and reduced dosing frequency. In anexperiment it was found that the placement ofone, two or three ribonucleotides at the 50 end ofthe modified siRNA of HBV263 and HBV1583improved the median inhibitory concentration ofthe stabilized siRNAs by approximately fivefold.
5.6.4 Bioconjugation
A siRNA can be linked to a carrier molecule orcovalently conjugated to a targeting ligand via acleavable spacer to increase its cellular uptake orconfer cell-specific targeting. To date, siRNAhas been conjugated to lipids, polymers, pep-tides, and aptamers. Conjugation of siRNA toapatamer which can bind to prostate-specificmembrane antigen (PSMA) has shown somepromise for targeted delivery of siRNA toprostate cancer. siRNAs have also been conju-gated to lipids like lithocholic and lauric acids atthe 50-end of the sense strand and showedincreased cellular uptake of siRNAs in humanliver cells without the use of any transfectionreagent. Conjugated cholesterol to the 30-end ofthe sense strand of siRNA targeting ApoB, anessential protein for assembly and secretion ofvery-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), has been formed thatdemonstrate significant increase in the levels ofthis conjugate in the liver.
5.6.5 Nanoparticle Delivery
The generation of nanosized particles is beinginvestigated to enhance the delivery of siRNAbased drugs and to circumvent the limitations ofdifficult to transfect cells such as lymphocytes.Numerous targeted nanoparticles have been usedto overcome one or more of the barriersencountered by siRNA when trafficked to thecytosol. Antibody-protamine fusion bodies andantibody-targeted liposomes have been usedsuccessfully for primary lymphocytes to targethuman integrin lymphocyte function associatedantigen-1 (LFA-1). Calando PharmaceuticalsInc. has developed a cationic cyclodextrin-con-taining polymer that binds siRNAs for systemicdelivery. Cyclodextrin containing polycationbinds to the anionic backbone of the siRNA andthen self-assemble into nanoparticles ofapproximately 50 nm in diameter.
5 RNA Interference and its Applications 63
5.6.6 Hydrodynamic Injection
Quick injection of siRNA in a large volume ofphysiological buffer effectively localizes duplexsiRNA in the liver. In rats, administration of aVEGF (vascular endothelial growth factor)—specific siRNA resulted in more than 75 %inhibition of pathological neovascularization.Due to the invasiveness of the injection tech-nique, hydrodynamics-based transfection is notappropriate for clinical applications at this point.However, recent advances in using a computer-controlled, catheter-guided injection device havegreatly improved the precision and reproduc-ibility of this approach. To date, the device hasonly been used for the delivery of DNA, butsiRNA should be equally applicable in thisapproach.
5.6.7 Direct Administrationinto Target Organs
For dsRNA delivery several direct methods canbe applied. Electroporation is used in simplerorganisms, whereas microinjection of dsRNAinto germ line or early embryo is preferred inmulticellular organisms. In C. elegans, injectioninto the intestine or pseudocoelom is almost asefficient as injection into germ line. Even feed-ing worms with bacteria that express dsRNA orsoaking worms in dsRNA solutions has beenapplied with some success. However, dsRNAinjected into early embryos is diluted upon celldivision; as a result early genes are more easilyinactivated than late genes, which is especially aproblem for higher organisms. In ongoing clin-ical trials of RNAi therapeutics, direct applica-tion of modified or unmodified siRNA into theeye (Allergen Inc. and Opko Health Inc.) andskin (TransDerm Inc.) through injection or byintranasal delivery (Alnylam) are the leadingmethods of administration. However, thesedelivery methods may be limited to a few indi-cations in a small number of accessible organssuch as the lung, eye and skin.
5.6.8 Delivery into Plants
siRNAs have been delivered into tobacco plantsby biolistic pressure to cause silencing of GFPexpression. Silencing occasionally was detectedas early as a day after bombardment, and itcontinued to potentiate up to 3–4 days post-bombardment. Systemic spread of silencingoccurred 2 weeks later to manifest in the vas-cular tissues of the non-bombarded leaves thatwere closest to the bombarded ones. After amonth or so, the loss of GFP expression wasseen in nonvascular tissues as well. RNA blothybridization with systemic leaves indicated thatthe biolistically delivered siRNAs induced thede novo formation of siRNAs, which accumu-lated to cause systemic silencing.
5.7 Applications of RNAi
The new technique was taken up by lots ofenthusiasm by scientists. It was soon realizedthat there has not been a tool this sharp in lastmany years. Besides being an area of intense,upfront basic research, the RNAi process holdsthe key to future technological applications.
5.7.1 Gene Knockdown
RNAi is a specific, potent, and highly successfulapproach for loss-of-function studies in virtuallyall eukaryotic organisms. As RNAi relies on thesequence-specific interaction between siRNAand mRNA, siRNAs can be tailored to silencealmost any gene. Using this mechanism,researchers can cause a drastic decrease in theexpression of a targeted gene. Studying theeffects of this decrease can show the physio-logical role of the gene product. Since RNAimay not totally abolish expression of the gene,hence it is more appropriate to call it ‘‘knock-down’’ to differentiate it from ‘‘knockout’’ pro-cedures in which expression of a gene is totallyextinguished.
64 J. Saxena
Genome sequencing projects generate awealth of information. The ultimate goal of suchprojects is to accelerate the identification of thebiological function of genes. The function ofgenes can be analyzed with an appropriate assay,by examining the phenotype of organisms thatcontain mutations in the gene, or on the basis ofknowledge gained from the study of relatedgenes in other organisms. However, a significantfraction of genes identified by the sequencingprojects are new and cannot be rapidly assignedfunctions by these conventional methods. RNAitechnique has come to rescue as it is not onlyaccurate but can also be used on a breathtakingscale. Although RNAi is unlikely to replace theexisting knockout technology, it may have atremendous impact for those organisms that arenot amenable to the knockout strategy.
The relative ease with which genes can besilenced using RNAi has caused a minor revo-lution in molecular biology. siRNAs that havebeen chemically synthesized or created byin vitro transcription systems, can inducesilencing in several organisms, including mam-malian cells. In the laboratory, this techniqueseems to be rapid and convenient, and can beused to tackle many genes at the same time. Asdiscussed earlier it can be achieved remarkablyeasily in C. elegans as gene silencing occurs bydirect feeding on bacteria which were earlier fedwith dsRNA that expressed it or even soakingworms in dsRNA. The effect was found to betransmitted to the next generation.
Extensive efforts in bioinformatics have beendirected toward the design of successful dsRNAreagents that maximize gene knockdown butminimize ‘‘off-target’’ effects, which arise whenan introduced RNA has a base sequence that canpair with, thus reduce the expression of multiplegenes at a time. Such problems occur morefrequently when the dsRNA contains repetitivesequences. A multitude of software tools havebeen developed for the design of general mam-mal-specific and virus-specific siRNAs that areautomatically checked for possible cross-reactivity.
Although long strand of RNA designed to becleaved by Dicer can also be used but in most
mammalian cells, shorter RNAs are preferred sothat induction of mammalian interferon responsecan be avoided. Specialized laboratory tech-niques have also been developed to improve theutility of RNAi in mammalian systems byavoiding the direct introduction of siRNA, forexample, by stable transfection with a plasmidencoding the appropriate sequence from whichsiRNAs can be transcribed, or by more elaboratelentiviral vector systems allowing the inducibleactivation or deactivation of transcription,known as conditional RNAi. Julie Ahringer’sgroup at the University of Cambridge has cre-ated a library of more than 16,000 cloned dsR-NAs (around 86 % of the C. elegans genome).By feeding these clones to worms, they havedetermined the function of 1,722 genes, most ofwhich were previously unknown. The researchteam of Gary Ruvkun at Harvard MedicalSchool has identified many hundreds of genesinvolved in fat storage. Similarly, GregoryHannon’s group at Cold Spring Harbor is look-ing to determine the functions of every gene inthe human genome. The list of projects in thisarea is seemingly endless. In another studyconducted by Mutti et al. (2006), microinjectionof long dsRNA was shown to transiently silencethe expression of two marker genes in the peaaphid, Acyrthosiphon pisum. They found RNAiknockdown of a salivary transcript leading tolethality.
5.7.2 Therapeutic Possibilities
Given the gene-specific features of RNAi, it isconceivable that this method will play animportant role in therapeutic applications. Thisapproach relies on designing the dsRNA fromthe DNA sequence of a gene that can specificallyand effectively silence a disease-related gene.There was an initial problem in developingtherapeutic approaches as dsRNAs that are 30nucleotides in length or longer can trigger non-sequence-specific interferon responses in mam-malian cells. But this has been overcome bydelivering siRNAs into mammalian cells inculture. Tremendous amount of work is going on
5 RNA Interference and its Applications 65
in this direction throughout the world on differ-ent diseases. Basically every human diseasecaused by activity from one or a few genesshould be amenable to RNAi based intervention.This list includes cancer, autoimmune diseases,genetic disorders, and viral infections.
5.7.2.1 Acquired ImmunodeficiencySyndrome
Acquired Immunodeficiency Syndrome (AIDS)causing HIV is first infectious agent targeted byRNAi because the life cycle and pattern of geneexpression of HIV is well understood. It wasdemonstrated by several scientists that siRNAscan inhibit HIV replication effectively in culture.HIV infection can also be blocked by targetingeither viral genes such as gag, rev, tat , and envor human genes viz. CD4, the principal receptorfor HIV. This is promising, as antiviral therapiesthat can attack multiple viral and cellular targetscould circumvent genetic resistance of HIV.Synthetic siRNAs have been used to target var-ious early and late HIV-encoded RNAs in celllines and in primary haematopoietic cellsincluding the TAR element 50, tat 51–53, rev51, 52, gag 54, vif 50, nef 50 and reversetranscriptase. These results have been achievedby transfecting the siRNAs into cells, but thechallenge lays in translating the same effectin vivo. However, the work is going on to makebetter delivery systems to circumvent the prob-lem, many groups have designed promoter sys-tems that can express functional siRNAs whentransfected into human cells. Early results haveshown that this can decrease replication of HIVconsiderably.
5.7.2.2 HepatitisHepatitis viral infection is one of the leadingcauses of liver cirrhosis and hepato cellularcarcinoma (HCC) and remains a challenge formodern medicine. Current treatment strategiesof Hepatitis B virus (HBV) infection includingthe use of a-interferon (IFN) and nucleotideanalogues have met with only partial success.Therefore, it is necessary to develop more
effective antiviral therapies that can clear HBVinfection with fewer side effects. Early RNAistudies noted that RNA silencing was prominentin the liver, which made this organ an attractivetarget for therapeutic approaches. Hepatitiscaused by HBV and Hepatitis C virus (HCV) hasbeen an important target for potential RNAitherapy. The first in vivo demonstration of RNAiefficacy against a virus implicated a hydrody-namic co-delivery of an HBV replicon and anexpression unit encoding an anti-HBV siRNA inmice. This study with siRNA showed a signifi-cant 99 % knockdown of HBV core antigen inliver hepatocytes providing an important proofof principle for future antiviral application ofRNAi in liver.
Many immune-related liver diseases arecharacterized by apoptosis, which is mediatedby a protein called Fas. Judy Lieberman’s groupinjected siRNA targeting Fas intravenously intotwo models of autoimmune hepatitis in mice.This decreased Fas mRNA and protein levels inhepatocytes and protected the cells against liverinjury from apoptosis, even when siRNA wasadministered after the induction of injury.Hence, the successful use of chemically syn-thesized siRNA, endogenous expression of smallhairpin RNA (shRNA) or miRNA to silence thetarget gene make this technology towards apotentially rational therapeutics for HBVinfection.
5.7.2.3 CancerThe potential for using RNAi to treat metastaticcancers depends on finding good cellular targets.With respect to future medical applications,siRNA-based therapy seems to have a greatpotential to combat carcinomas, myeloma andcancer caused by over expression of an onco-protein or generation of an oncoprotein bychromosomal translocation and point mutations.Gregory Hannon and colleagues have usedRNAi to silence expression of p53, the ‘guardianof the genome’ which protects against anytumor-associated DNA damage by introducingseveral p53-targeting short hairpin RNAs(shRNA) into stem cells in mice. The shRNAs
66 J. Saxena
produced a wide range of clinical effects, rang-ing from benign to malignant tumors, theseverity and type of which correlated with theextent to which the shRNA had silenced p53. Astumor suppressors such as p53 usually work aspart of a complex and finely regulated network,the ability to dampen these networks to varyingdegrees will be of enormous value when itcomes to investigating the early stages of dis-ease. The success of these modified stem cellsalso gives hope that this could treat diseases inwhich stem cells can be modified ex vivo andthen re-introduced into the affected individual.
In chronic myelogenous leukemia (CML),RNAi has been used to target genes expressingoncogenic fusion proteins such as Brc-Abloncoprotein p210. The feasibility of delivery ofsiRNAs and expressing siRNAs viral vectors todiseased regions of the brain coupled withselective targeting of SNPs in the mutant tran-scripts may be promising for using it as an anti-cancer therapeutic agent. Transfected siRNAsachieve significant gene knockdown for3–7 days before its natural degradation. Thismay not be sufficient for therapy but can beuseful for studying the immediate effects ofinhibiting gene expression for drug targets ordrug target validation.
Researchers at the Charity Cancer Research,UK, and the Netherlands Cancer Institute areworking to generate a large library of humancells, each containing a silenced gene with agoal of silencing 300–8,000 cancer genes ini-tially, and then eventually covering the entirehuman genome. This will help to uncover all thegenes that become over expressed in humancancers and to find out precisely what needs tobe taken away from a cancerous cell in order tomake it normal again.
5.7.2.4 MalariaMalaria, a mosquito-borne infectious disease ofhumans and animals, is a leading cause of mor-bidity and mortality worldwide. Recent evidencestrongly suggests that RNAi can play a key role inidentifying new genetic means of controlling
mosquito-borne diseases. Anopheles mosquitoeswere transfected with a transgene containing twocopies of the target gene arranged in an invertedrepeat configuration. Hairpin RNA is expressedin vivo whenever the inverted repeat is tran-scribed from an upstream promoter. By placingdsRNA expression under the control of a tissue-specific and time-specific promoter, dsRNAexpression can be tailored to coincide spatiallyand temporally with the journey of the parasitethrough the mosquito. Both parasite receptors andimmune components protective of the parasite areputative targets for engineering parasite resis-tance through RNAi and, in principle, mosquitostrains that have been rendered refractory tomalaria transmission could be released in the fieldto replace wild type, permissive populations andachieve malaria eradication (Angaji et al. 2010).
5.7.2.5 Ocular DiseasesSeveral diseases affect the eye, either directly oras part of a system-wide problem. Wallach(2004) illustrated that the enclosed nature of eyemakes ocular diseases ideal targets for siRNA-based therapies. siRNA was detected for at least5 days when injected into the vitreous cavity.The sequence specificity of siRNA resulting intargeting of a single gene combined with localadministration in the relatively isolated confinesof the eye provides an ideal way to study eye-specific effects of gene disruption.
The first ever clinical trial in man in 2004 byAcuity Pharmaceuticals have been directed atthe treatment of age-related macular degenera-tion (AMD), which causes blindness or limitedvision in millions of adults annually in patients.A siRNA targeting the vascular endothelialgrowth factor (VEGF), a primary cause ofovergrowth of blood vessels in the ‘wet’ form ofAMD, was administered by intravitreal injec-tion. The primary goal of this phase 1 study wasto evaluate the safety of the siRNA. A singleintravitreal dose of Sirna-027, ranging from100–800 lg, appears to be safe and well toler-ated with no systemic or local adverse eventsrelated to the drug.
5 RNA Interference and its Applications 67
5.7.2.6 Metabolic DiseasesRNAi holds considerable promise as a thera-peutic approach to silence disease causinggenes, particularly those that encode so-called‘nondrugable’ targets. The endogenous genesinvolved in the cause or pathway of metabolicdiseases are silenced. Besides, no toxicity andadverse effects are observed due to high potency,specificity, and chemical structure of RNAi. Afew studies utilized RNAi technology to targetkey genes involved in the regulation of gluco-neogenesis and provided in vivo proof-of-prin-ciple for the development of RNAi-basedtherapeutics for diabetes. A link between miR-NAs and diabetes seems increasingly likely. In2004, Poy and his group identified severalmiRNAs expressed selectively in pancreaticendocrine cell lines: among these was miR-375,over expression of which resulted in suppressedglucose-stimulated insulin secretion and whoseinhibition enhanced insulin secretion. Taniguchiet al. (2005) developed an adenovirus-mediatedRNAi technique that utilizes shRNAs to sub-stantially and stably reduce the expression ofinsulin receptor substrates IRS-1 and IRS-2specifically in the liver of mice to better under-stand the roles of these proteins in hepaticinsulin action. By knocking down IRS-1 andIRS-2 in the liver separately and together, theyshowed that IRS-1 signaling may be more clo-sely linked to the regulation of genes involved inglucose homeostasis, whereas IRS-2 signalingmay have specific roles in the regulation ofhepatic lipid metabolism.
Vector-based RNAi approach has been usedfor hyperglycemia through phosphoenol pyru-vate carboxykinase (PEPCK), a rate-controllingenzyme in gluconeogenesis. RNAi also holdspromise for the development of novel thera-peutic strategies for disorders that are yet diffi-cult to treat such as obesity, neuropathic painand depression. Although development ofRNAi-based therapeutics is in its infancy, earlyclinical studies show the use of this new class oftherapeutics to tackle metabolic diseases.
5.7.2.7 Neurodegenerative DisordersNeurodegenerative disease is an umbrella termfor a range of conditions which primarily affectthe neurons in the human brain. The diseasessuch as Alzheimer’s, Parkinson’s, Huntington’sand amyotrophic lateral sclerosis (ALS) areexamples of relatively common age-relatedneurodegenerative disorders that are increasingas average life expectancy increases. Each dis-order is characterized by the dysfunction anddeath of specific populations of neurons. It isbelieved that specific genetic mutations areresponsible for these diseases. Recent studieshave shown that cultured neurons can be effi-ciently transfected with siRNAs to effectivelysilence the target genes.
5.7.2.8 Cardiovascular and CerebralVascular Diseases
Cardiovascular diseases are the leading cause ofdeath in developed countries. siRNA is a recentadvancement that provides the possibility ofreducing gene expression at the post-transcrip-tional level in cultured mammalian cells. Thistechnology has been exploited in the process ofatherosclerosis (progressive occlusion of arter-ies) and also to reduce the damage to heart tissueand brain cells that patients suffer following amyocardial infarction or stroke.
5.7.2.9 Genetic DiseasesA genetic disorder is an illness caused byabnormalities in genes or chromosomes, espe-cially a condition that is present from birth.siRNA seems to be a good candidate for thetreatment of genetic diseases because the target-specific nature of siRNA may be able to target asingle mutation in the genome, which is causingthe disease. Once the target has been located,and the siRNA has been effectively delivered, ithas the potential to down-regulate the singlemutation, which could restore proper health. Thechallenge in this method is finding the optimal
68 J. Saxena
delivery method for specific sites in the genome,while not affecting the normal genes in the cell.Each genetic disease causes an unique mutation,so finding the specific targets for each disease isthe limiting factor for the therapeutic potentialof siRNA. The use of siRNA for genetic treat-ment is also very expensive because of theunique sequence target and diversity amongindividuals, so customized solutions are needed.
5.7.2.10 Other DiseasesMany groups of researchers are involved instudies related to knocking down the diseasedgenes. Chi et al. (2003) reported that the siRNA-induced gene silencing of transient or stablyexpressed GFP mRNA was highly specific in thehuman embryonic kidney (HEK) 293 cellbackground. Among the first applications toreach clinical trials were the treatment of mus-cular degeneration and respiratory syncytialvirus (RSV). siRNAs were successfully used tosilence genes expressed from RSV and parain-fluenza virus (PIV), the RNA viruses that causesevere respiratory disease in neonates andinfants. Other proposed clinical trials use centeron antiviral therapies, including topical micro-bicide treatments that use RNAi to treat infec-tion by herpes simplex virus type 2, silencing ofinfluenza gene expression and inhibition ofmeasles viral replication.
5.7.3 Functional Genomics
Functional genomics is the field that attempts todescribe gene functions and interactions. Therehas been a lack of powerful tools for systematicanalysis of mammalian gene function, but RNAimay now provide such a strategy. Genomes formany organisms including human has alreadybeen sequenced and this technique provides away by which this enormous wealth of infor-mation can be translated into functional defini-tions for every gene. It may be useful to analyzequickly the functions of a number of genes in awide variety of organisms. Most functionalgenomics applications of RNAi in animals have
used common model organisms such as C. ele-gans and Drosophila melanogaster. C. elegansis particularly useful for RNAi research becauseof the heritable effects of the gene silencing andsimple delivery of the dsRNA. Chromosomes Iand III of C. elegans have been screened byRNAi to identify the genes involved in celldivision and embryonic development. Recently,a large-scale functional analysis of &19,427predicted genes of C. elegans was carried outwith RNAi. This study identified mutant phe-notypes for 1,722 genes. Another study per-formed a genome-wide screen to identify thegenetic components required for RNAi, using anengineered RNAi sensor strain of C. elegans.The RNAi screen identified 90 genes includingPiwi/PAZ proteins, DEAH helicase, RNAbinding/processing factors, chromatin-associ-ated factors, DNA recombination proteins,nuclear import/export factors, and 11 knowncomponents of RNAi machinery. Similarly, inD. melanogaster, RNAi technology has beensuccessfully applied to identify genes withessential roles in biochemical signaling cas-cades, embryonic development, and other basiccellular processes. With the recent developmentof siRNA and shRNA expression libraries, theapplication of RNAi technology to assign func-tion to cancer genes and to delineate molecularpathways in which these genes affect in normaland transformed cells, are expected to contributesignificantly to the knowledge necessary todevelop new techniques and also to improveexisting cancer therapy.
Stable transcription of RNAi triggered fromsuitable expression cassettes integrated into thehost cell genome by viral gene transfer caninduce long-term and heritable gene silencing inmammalian cells. However, the use of RNAi asa genetic tool is limited due to difficulties inidentifying efficient RNAi triggers, the problemof effective delivery and off-target effects, aswell as potential genotoxic side effects of viralgene transfer strategies.
Functional genomics using RNAi is a partic-ularly attractive technique for genomic mappingand annotation in plants because many plants arepolyploid, which presents substantial challenges
5 RNA Interference and its Applications 69
for more traditional genetic engineering meth-ods. For example, RNAi has been successfullyused for functional genomics studies in hexa-ploid bread wheat as well as Arabidopsis andmaize, common plant model systems. Plantnematology in the genomics era is now facing thechallenge to develop RNAi screens adequate forhigh-throughput functional analyses. RNAi-mediated suppression of genes essential fornematode development, survival, or parasitism isrevealing new targets for nematode control.
5.8 Conclusion
RNAi is a mechanism for controlling normalgene expression. Lately, it has become a pow-erful tool for gene therapy for many diseases,besides being used for probing gene functions,making transgenic plants, and rationalizing drugdesign. However, further refinements arerequired for the development of a delivery sys-tem with little or no toxicity, but with high effi-ciency and selectivity for consistent and stableexpression levels before it can be used success-fully. Since human subjects are involved in genetherapy, hence it is also important to perform arisk–benefit analysis and considering the risks ofgenerating infection-competent viruses or intro-ducing genetic changes in germ line cells.
References
Agarwal A, Dasaradhi PVN, Mohmmed A, Malhotra P,Bhatnagar RK, Mukherjee SK (2003) RNA interfer-ence: biology, mechanism, and applications. Micro-biol Mol Biol Rev 67(4):657–685
Angaji SA, Hedayati SS, Poor RH, Madani S, Poor SS,Panahi S (2010) Application of RNA interference intreating human diseases. J Gen 89:527–537
Bernstein E, Caudy A, Hammond S, Hannon G (2001)Role for a bidentate ribonuclease in the initiation stepof RNA interference. Nature 409(6818):363–366
Chi JT, Chang HY, Wang NN, Chang DS, Dunthy N,Brown PO (2003) Genomewide view of gene silenc-ing by small interfering RNAs. Proc Natl Acad SciUSA 100:6343–6346
de Jonge J, Holtrop M, Wilschut J, Huckriede A (2006)Rreconstituted influenza virus envelope as an efficientcarrier system for the cellular delivery of smallinterfering RNAs. Gene Ther 13(5):400–411
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE,Mello CC (1998) Potent and specific genetic inter-ference by double-stranded RNA in Caenorhabditiselegans. Nature 391:806–811
Hammond SM, Boettcher S, Caudy AA, Kobayashi R,Hannon GJ (2001) Argonaute2, a link betweengenetic and biochemical analyses of RNAi. Science293:1146–1150
Lee RC, Feinbaum RL, Ambros V (1993) The C. elegansheterochronic gene lin-4 encodes small RNAs withantisense complementarity to lin-140 0. Cell 75(5):843–854
Mutti NS, Park Y, Reese JC, Reeck GR (2006) RNAiknockdown of a salivary transcript leading to lethalityin the pea aphid, Acyrthosiphon pisum. J Ins Sci 6:38
Napoli C, Lemieux C, Jorgensen R (1990) Introductionof a chimeric chalcone synthase gene into petuniaresults in reversible co-suppression of homologousgene in trans. Plant Cell 2:279–289
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Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X,Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N,Rorsman P, Stoffel M (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature432:226–230
Taniguchi CM, Ueki K, Kahn CR (2005) Complementaryroles of IRS-1 and IRS-2 in the hepatic regulation ofmetabolism. J Clin Invest 115:718–727
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70 J. Saxena
6DNA Microarray
Ashwini M. Charpe
Abstract
DNA microarray is a technology that has revolutionized the functionalgenomics with a wide array of applications. It is an arrangement of a largenumber of known genes or corresponding cDNA probes, for a givenphysiological condition of any living being; that are placed precisely as dotson a glass slide or chip or coated on beads. It works on the principle ofSouthern hybridization wherein DNA is hybridized with DNA to confirmthe expression of a gene. The only difference is, in microarrays probes areplaced on solid surface and test DNA is in the hybridization solution, whichis just opposite to Southern hybridization where DNA to be diagnosed isplaced on nylon or nitrocellulose membrane and probe is in thehybridization solution. DNA microarray helps in screening of thousandsof genes in one go to understand their expression in a given physiologicalcondition, when hybridized with the test DNA. During hybridization,fluorescent dyes attached to probes produce emissions of specific colorbased on complete partial and no binding of DNA to the probes. Afterhybridization these emissions can be observed and recorded under aconfocal laser microscope and further analyzed with the help of imageanalysis software to understand set of genes up or down regulated in the testDNA and to determine fluorescence intensities that allow the quantitativecomparison between the two test DNAs for all genes on the array. Thistechnique is useful in gene expression profiling, comparative genomichybridization, checking GeneID, SNP detection, alternative splicingdetection, fusion gene detection and genome tilling to empirically detectexpression of transcripts, or alternative splice forms. It has been widelyapplied in studies related to cancer biology, microbiology, plant science,environmental science, etc.
6.1 Introduction
A microarray is a 2D arrangement of knownbiochemical or chemical constituent of a bio-logical system on a solid substrate like glass
A. M. Charpe (&)Plant Pathology Section, College of Agriculture,Dr.Panjabrao Deshmukh Krishi Vidyapeeth, Akola,Maharashtra 444104, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_6, � Springer India 2014
71
slide or silicon chip that is useful in highthroughput screening of biological material.Microarray methods were initially developed tostudy differential gene expression using complexpopulations of RNA. Refinements of thesemethods now permit the analysis of copy num-ber imbalances and gene amplification ofDNA and have recently been applied to thesystematic analysis of expression at the proteinlevel. Many of the guiding principles of globalanalysis using microarrays are, in principle,applicable at the RNA, DNA or protein level.Till date, various types of microarrays usingavailable biochemical information have beendeveloped that are as following (http://en.wikipedia.org/wiki/Microarray):• DNA microarrays: cDNA microarrays, oligo-
nucleotide microarrays, and SNP microarrays.• MMChips: for surveillance of microRNA
populations.• Protein microarrays.• Tissue microarrays.• Cellular microarrays or Transfection
microarray.• Chemical compound microarrays.• Antibody microarrays• Carbohydrate arrays or glycoarrays.
This chapter deals with the DNA microarrayswhich are recognized as multiplex technologythat either measure DNA or use DNA as part ofits detection system. DNA microarrays are pre-pared by robotic plotting of picomoles(10-12 mol) of specific DNA sequences knownas probes or reporters on a solid substrate likeglass slide, silicon chip, Affymatrix chip (Affychips in short), and microscopic beads calledIllumina. In standard microarrays, the probes areattached to the respective solid surface by sur-face engineering using epoxy-silane, amino-silane, lysine, polyacrylamide or others thatbinds the probe to the surface by a covalentbond. With this technique, thousands of probescan be fixed to each cm2 area on the solid sur-face appearing like tiny dots known as features.An array can contain tens of thousands ofprobes. These probes might be prepared from-• Available cDNA sequences from cDNA
libraries developed by trapping expressed
sequence tags (ESTs), those are usually200–300 bp long;
• By in silico synthesis of 20–30 bp long non-overlapping oligonucleotide sequences; and
• By using the nucleic acid sequences distin-guishing the single nucleotide polymorphism(SNPs).The DNA microarray is hybridized with a
cDNA or cRNA sample known as target and isprepared from a biological system under study,which is subjected to a specific condition, underhighly stringent situations. For detection andquantification of hybridization of probe with thetarget, the target is labeled with the signal pro-ducing fluorophore, silver, or chemiluminescencelabels. These signals help in determining the rel-ative abundance of nucleic acid sequences in thetarget. Likewise, DNA microarray facilitates themeasurement of expression levels, detection ofSNPs and genotyping or resequencing of mutantgenomes (http://en.wikipedia.org/wiki/DNA_microarray).
6.2 History
DNA microarrays were gradually developedfrom southern blotting (Maskos and Southern1992). Hybridization of probe with the blottednucleic acid fragment results in separation ofradioactivity or chromogenic substrate that canbe detected by autoradiography or colorimeter.
In the first step DNA microarray was preparedfrom lysed bacterial colonies having differentgene sequences. A total of 378 such colonies werearranged in an array and hybridized with the DNAof normal and tumor tissue in replications (Au-genlicht and Kobrin 1982) to study the geneexpressions. In the second step, 4,000 humangene sequences were arranged in an array andhybridized with the DNA preparations fromhuman colonic tumors and normal tissue, theresults were processed by computer aided scan-ning and image processing for quantitative anal-ysis of the expressed sequences. This array wasalso used for comparison of colonic tissues atdifferent genetic risk (Augenlicht et al. 1987,1991). In the third step, in 1987, a collection of
72 A. M. Charpe
distinct DNAs was used for preparation of anarray and was used for expression profilingleading to identification of genes whose expres-sion was modulated by interferon (Kulesh et al.1987). Initially, these gene arrays were preparedby spotting cDNAs onto filter paper with a pin-spotting device. In the fourth step, miniaturizedmicroarrays were used for gene expression pro-filing in the year 1995 (Schena et al. 1995). Thatled to the preparation of microarray havingcomplete genome of Saccharomyces cerevisiae,in 1997 (Lashkari et al. 1997).
6.3 Principle
DNA Microarrays are based on the principle ofSouthern blotting where fragmented DNA isattached to a solid substrate like nylon or nitro-cellulose membrane and then hybridized with thechemiluminiscence or radioactively labeledprobe prepared from a known gene or DNAfragment under stringent conditions. Thehybridization between two DNA strands takesplace by the property of complementary nucleicacid sequences to specifically pair with eachother by forming hydrogen bonds betweencomplementary nucleotide base pairs. A highnumber of complementary base pairs in anucleotide sequence results in tighter noncova-lent bonds between the two strands. Afterwashing-off of nonspecific bonding sequences,only strongly paired strands remain hybridized.In DNA microarrays, this situation is slightlyaltered wherein, thousands of nonlabeled probes
are blotted on the solid surface and these chipsare subjected to hybridization with the fluores-cently labeled target sequence, i.e., sample DNA(Fig. 6.1). On binding of fluorescently labeledtarget sequence to a probe sequence, a signal isgenerated that depends on the strength of thehybridization determined by the number ofpaired bases, the hybridization conditions (suchas temperature), and washing after hybridization.Total strength of the signal, from a spot (called asfeature), depends upon the amount of targetsample binding to the probes present on that spot.Microarrays use relative quantification in whichthe intensity of a feature is compared to theintensity of the same feature under a differentcondition, and the identity of the feature isknown by its position. The steps involved inmicroarray experiment are elaborated in Fig. 6.2.
6.4 Types and Fabrication of DNAMicroarrays
Broadly DNA arrays are classified as solidphase, beaded and electronic arrays.
6.4.1 Solid-Phase Arrays
The traditional solid-phase array is a collectionof orderly microscopic spots (features); eachwith a specific probe and attached to a solidsurface, such as glass, plastic or silicon biochipcommonly known as a genome chip, DNA chip,or gene array. Thousands of such features can be
Fig. 6.1 Hybridization ofthe target to the probe inDNA microarray (Sourcehttp://en.wikipedia.org/wiki/DNA_microarray)
6 DNA Microarray 73
placed on known locations on a single DNAmicroarray chip (http://en.wikipedia.org/wiki/DNA_microarray). On the basis of informationused for the probes the solid phase DNAmicroarrays can be of two types, Printedmicroarrays (oligonucleotide based microarrays)and GeneChips (in situ synthesized oligonu-cleotide microarrays).
6.4.1.1 Oligonucleotide-Based PrintedMicroarrays
The first DNA arrays used for research workwere oligonucleotide-based printed microarrays.These microarrays require either PCR amplified(ds-DNA) or synthesized oligonucleotides (ss-DNA) of known gene sequences as probes. Inthese microarrays, glass slides are used as solidsurface for spotting or printing of the probes.Cheung et al. (1999) observed that glass slidesare not only observed to be stable during thevaried experimental situations but also produceleast background noise during hybridization,thus yielding clearer images. Probes can beprinted in two ways by contact printing or bynoncontact printing. Whichever the method ofprinting is used in both the cases an array of100–150 lm features is created by applying fewnanolitres of probe solution per spot. Contactprinting means direct application of probesolution on the solid surface with the help ofprint pins, whereas in noncontact printing asmall droplets of probe solution is placed on thesolid surface by the print head. Printing is doneprecisely so as to avoid mixing of adjacentfeatures. In comparison to GeneChip and highdensity bead arrays, oligonucleotide-basedprinted arrays have low density of probes
ranging from 10,000 to 30,000 features per arraydue to the relatively large size of the features init but has more features than suspension beadarrays and electronic microarrays.
When dsDNA is used as probes, it is requiredto denature them either at the time of printing orafter spotting on the solid surface so that theybecome available for the hybridization (Tomiukand Hofmann 2001). For getting the probesattached on the glass surface, two methods areused, first, either slides are applied with a posi-tively charged coating that allows the electro-static interaction with the negative charge of thephosphate backbone of the DNA (Ehrenreich2006) and second, by UV-cross-linking of aminegroups present on treated slides with the cova-lent bonds present between the thymidine basesin the DNA (Cheung et al. 1999). Each dsDNAprobe used in these arrays represents a differentgene and is usually 200–800 bp long. Theseprobes should have high specificity and shouldbe free from contaminants so that there is nointerference while hybridization. Generally,these probes have a high sensitivity but theysuffer in specificity and might be contaminatedwith nonspecific amplicons (Hager 2006). Toselect for the probe specificity, amplicons can betested by sequencing or agarose gel electropho-resis and specificity of the hybridization data canbe improved by including multiple gene seg-ments among the probes so as to workout theredundancy. At times decreased specificity isadvantageous when the genomic sequence underanalysis has high natural polymorphism,although it does not help when we have to dif-ferentiate among highly similar target sequen-ces, therefore they are not useful as far as theclinical diagnostics is concerned. At the same
Fig. 6.2 The steps involved in a microarray experiment (Source http://en.wikipedia.org/wiki/DNA_microarray)
74 A. M. Charpe
time, use of longer probes results in highermelting temperatures and mismatch tolerancethat decreases the specificity of the probes due torandom hybridization to nontarget sequences,hence for any microarray experiment probelength should be chosen carefully.
In oligonucleotide microarrays with ssDNAprobes, probes are chemically synthesized shortsequences usually ranging from 25–80 bp inlength, in contrast to gene expression microarrayswhere they may be up to a length of 150 bp (Chouet al. 2004). In these microarrays due to theshorter length of probes, fewer errors areobserved during probe synthesis and they help instudies focusing small genomic regions likepolymorphisms. Another benefit due to the smallprobe length is increased specificity while tar-geting specific genomic regions although itreduces the sensitivity of detections. It isobserved that the sensitivity and the strength ofhybridization signal increases with the length ofthe probe used. In case of very short oligonucle-otides, addition of spacers or application of ahigher concentration of probe during printingimproves the strength of hybridization signal(Chou et al. 2004). Ramdas et al. (2004) observedeightfold increase in sensitivity when 70 meroligonucleotides were used as probes as com-pared to 30 mer probes for the genes having lowlevels of expression. ssDNA oligonucleotideprobes are easy to manufacture but care isrequired that all the probes on the array shouldhave melting temperature lying within a range of5 �C and should not have palindromic sequences.
Hence, it is required to test the probes in advanceso that the hybridization data generated throughany experiment using ssDNA probes remainunbiased (Chou et al. 2004). Attachment ofssDNA probes to glass surface is facilitated due tocovalent linkage between modified 50 or 30 endshaving amino group with the aldehyde or epoxyfunctional groups coated on the slides. Oligonu-cleotide microarrays manufactured by RocheNimbleGen (Madison, WI) and Agilent Tech-nologies (Palo Alto, CA) are good examples.Each NimbleGen microarray contain [106 fea-tures. The available formats are 1 9 2.1 millionfeatures, 3 9 720,000 features, 1 9 385,000features, 4 9 72,000 features, and 12 9 135,000features per slide. Agilent microarrays are avail-able in the formats having 1 9 244,000 features,2 9 105,000 features, 4 9 44,000 features, and8 9 15,000 features per slide (Miller and Tang2009). An oligo microarray with approximately40,000 probes is shown in Fig. 6.3 to have an ideaof how it looks after hybridization.
6.4.1.2 GeneChips (in situ SynthesizedOligonucleotide Microarrays)
GeneChips are produced by in situ synthesis ofoligonucleotides directly onto quartz or glasswafers having a size of 1.2 cm2. Tens of thou-sands of oligonucleotides are placed on onewafer by synthesizing one nucleotide at a time.Each oligonucleotide probe on the array repre-sents one gene. Length of the probes used inthese arrays depends on the purpose for which
Fig. 6.3 Example of anapproximately 40,000probe spotted oligomicroarray with enlargedinset to show details(Sourcehttp://en.wikipedia.org/wiki/DNA_microarray)
6 DNA Microarray 75
these microarrays are designed. GeneChipsdesigned by Agilent technology use longerprobes having 60 bp lengths, whereas in Af-fymatrix GeneChips shorter probes having alength of 25 bp are used. Typically, GeneChipshave [106 features per microarray that actuallydepends on the distance between the features(Weiszhausz et al. 2006).
For binding of the in situ synthesized probesto the solid surface mainly a modification ofsemiconductor photolithography technology isused. In this technique, quartz surface of theGeneChip is coated with synthetic linkersmodified with light-sensitive protecting groups(Fodor et al. 1991). Due to this chemical pro-tection, addition of nucleotide to the microarraysurface is restricted till its deprotection by UVlight. This means nucleotides modified with aphotosensitive protecting group can be added togrowing oligonucleotide chains by exposing thearray surface to UV light. Further, photolitho-graphic masks are used for directing the specific
nucleotides to exact probe sites. Photolitho-graphic mask has a defined pattern of windows;those selectively transmit or block the UV lightfor the targeted feature on the microarray surfacethat is chemically protected. Due to this kind offiltration, an area of the microarray surface thatdoes not receive UV light remains protected anddoes not allow addition of nucleotides, whereasareas exposed to UV light gets deprotected, andspecific nucleotides can be added to these sites.Likewise, the order of nucleotide addition isdirected by the pattern of windows in eachphotolithographic mask. Therefore, in situ probesynthesis requires cycles of masking, UVexposure, and addition of nucleotide bases(either A, C, T, or G) to the growing oligonu-cleotide chain on the solid surface (Fig. 6.4)(Weiszhausz et al. 2006). In addition to arraysynthesis using photolithography mask Nimble-Gen Systems has developed a technique ofmaskless array synthesis that provides flexibilityof use with large number of probes required in
3. Coupling
Binding of complementary
nucleotide on the target feature
2. Activation
The target features getexposed for binding of
nucleotides
1. Light deprotection UV irradiation of target features on slide surface
by photolithographic mask
changed in Mask is
every cycle as per targets to
be exposed
Fig. 6.4 Photolithographic production of oligonucleotide microarrays
76 A. M. Charpe
in situ synthesized oligonucleotide arrays toimprove sensitivity, specificity, and statisticalaccuracy due to shorter probe lengths.
The main advantage of GeneChips is theirability to measure the absolute expression ofgenes in cells or tissues. Their disadvantages aretheir higher costs and current inability tosimultaneously compare, on the same array, thedegree of expression of two related biologicalsamples.
For expression analysis using oligonucleotidemicroarray hybridization (Fig. 6.5), firstlylabeled RNA samples are prepared by convert-ing extracted mRNA to double-stranded cDNA.The cDNA is then copied to antisense RNA(cRNA) by in vitro transcription reaction per-formed in presence of biotin-labeled ribonucle-otide triphosphates (UTP or CTP). FragmentedcRNA (50–100 nucleotides) is used for hybrid-ization. After a brief washing step to removeunhybridized cRNA, the microarrays are stainedby streptavidin phycoerythrin and scanned(Aharoni and Vorst 2001).
Both types of oligonucleotide microarraysproduce cleaner downstream hybridization data.
Reproducibility, simpler standardization, anduse of controls make these microarrays usefulfor clinical diagnostics. In comparison to in situ-synthesized microarrays, printed microarrayseems to be relatively simple and inexpensivewith flexibility to quickly adjust spotted probeson the basis of newer informations. However,in situ-synthesized microarrays are more robustdue to significant control measures included intheir design.
Now oligonucleotide arrays have also beendeveloped which combine the flexibilities andqualitative advantages of synthetic probe arrayswith the benefits of simultaneous analysis possi-ble by spotted glass array. Further, custom-designed microarrays have also been madeavailable by Nimblegen and Agilent technologies.
6.4.2 Bead Arrays
The bead array is a collection of microscopicsilica/polystyrene beads, each with a specificprobe and a ratio of two or more dyes, which donot interfere with the fluorescent dyes used on
1.Collection oftissue sample for analysis
2. Isolation of mRNA
3. Preparation of double standed
cDNA by reverse transcription
4. In vitro transcription to prepare labeled antisense cRNA in presence of biotin labeled UTP or CTP
5. Fragmentation
6. Hybridization
7. Washing
8. Staining with fluorescent streptavidin
phycoerythrin stain
9. Scanning
Fig. 6.5 Steps involved in expression analysis using oligonucleotide microarrays hybridization
6 DNA Microarray 77
the target sequence. Bead arrays are mainly oftwo types, high density bead arrays and sus-pension bead arrays.
6.4.2.1 High-Density Bead ArraysIn high-density bead arrays silica beads of 3 lmsize are used as solid support for application ofprobes. After hybridization, these beads canrandomly self-assemble on the substrate. Twosubstrates available for this purpose are SentrixArray Matrix (SAM) and the Sentrix BeadChip.These arrays facilitate high density detection oftarget nucleic acids. One of the examples isIllumina bead arrays, San Diego, CA.
The SAM contains a bundle of 96 fiber-opticcables of 1.4 mm diameter. Each bundle repre-sents an array made up of 50,000 light-con-ducting fibers of 5 lm diameter; each of thisfiber has a microwell for assembling a singlebead (Fan et al. 2006). For example, in theUniversal Bead array, up to 1,536 bead types canbe accommodated. Here, every bead type rep-resents a unique gene sequence and when theyassemble on the fiber bundles 30 beads of eachtype becomes available for analysis in the array(Oliphant et al. 2002). Likewise, each SAMallows the analysis of 96 samples.
In Sentrix BeadChip up to 16 samples can beaccommodated on a silicon slide that has mi-crowells prepared for individual beads by mi-croelectromechanical systems technology (Fanet al. 2005). Gunderson (2009) has foundBeadChips more appropriate for very-high-densityapplications like whole-genome genotypingwhere 105–106 features are required for identi-fying genome-wide single nucleotide polymor-phisms (SNP).
Since the beads randomly assemble on thesubstrate, their location is not fixed and it isrequired to map their position by decodingprocess after every single application (Gunder-son et al. 2004). For the decoding purpose, eachbead type is covalently attached with 700,000copies of a unique capture sequence that acts asthe identifier of the bead (Kuhn et al. 2004). Forexample, in Universal BeadArrays such identi-fier sequences are called as IllumiCodes and
they are designed to specifically avoid homologywith human and mouse nucleic acid sequences.The mapping of these Illumina beads requires aseries of hybridization and rinse steps, so thatthe fluorescently labeled complementary oligo-nucleotides may bind to their specific beadsequence or IllumiCode and ultimately track thelocation of the corresponding bead type (Fanet al. 2006). This decoding process providesquality control for each feature used in themicroarray (Fan et al. 2005).
It is possible to process the SAM by using astandard microtiter plate that facilitates standardautomation and high-throughput processing.Similarly, individual arrays on the 16-sampleBeadChip are spaced as the standard multichan-nel pipettor for easy handling while use. 105–106
features can be supported by Bead arrays. Oneadvantage of bead arrays is their built-in redun-dancy that provides control for comparing datagenerated by more than one arrays. Further, dueto the uniqueness of each microarray by alteringthe bead pattern spatial bias can be identified.The work on developing the analysis tools forbead arrays is in progress (Miller and Tang2009). Bead arrays are found very useful instudies related to DNA methylation (Bibikovaand Fan 2009), profiling of gene expressions(Bibikova et al. 2004; Fan et al. 2004; Kuhn et al.2004), SNP detection, and in the InternationalHapMap Project (Gunderson 2009).
6.4.2.2 Suspension Bead ArraysIn suspension bead arrays, microscopic poly-styrene spheres, i.e., microspheres or beads areused as solid support for application of probesand flow cytometry technique is used fordetection of bead and target attached to it. Inthese arrays, beads remain suspended in thehybridization solution and do not require fiber-optic cables or silicon slides to immobilize thembefore scanning. All the previously discussedmicroarrays were two-dimensional, or planararrays whereas, suspension bead arrays arethree-dimensional arrays.
These arrays were first described by Horanand Wheeless (1977) for detections involving
78 A. M. Charpe
antigen and antibodies. Further, it was madepossible to detect multiple antibodies in onerun by using different sized microsphere sets(Scillian et al. 1989; McHugh et al. 1998). Insuspension bead arrays if 5.6 lm microspheresare filled with different concentrations of red(658 nm emission) and infrared (712 nm emis-sion) fluorochromes, each bead in the set of 100microspheres will have a distinct red to infraredratio providing each bead a unique spectraladdress. This provision has strengthened multi-ple detections using suspension bead arrays.When, microspheres with a specific spectraladdress is coupled with a specific probe, itbecomes equivalent to a feature of a two-dimensional microarray but when multiplemicrospheres with unique spectral addresses arecombined with multiple specific probes it ispossible to develop up to 100 combinations tostudy the target nucleic acids. Along with uniquespectral address and specific probe suspensionbeads are also provided with a fluorescentreporter that helps in detecting hybridization oftarget with the probe. Here, when hybridizationtakes place reporter separates to emit fluores-cence that can be detected by a flow cytometer.During flow cytometry, microsphere suspensionis passed through two lasers, i.e., 635 and532 nm. A 635 nm laser excites red and infraredfluorochromes and helps in detecting the uniquespectral address of beads and hence the identityof probe and target can be worked out, whereas,532 nm laser excites reporter fluorochromessuch as R-phycoerythrin and Alexa 532 andhelps in quantifying the amount of hybridizationtaken place on individual bead (Fig. 6.6).
Suspension bead arrays are used in followingthree ways for nucleic acid detections (Dunbar2006):1. Direct DNA hybridization: In this method,
PCR amplicons are directly hybridized toprobes hence it is known as direct DNAhybridization. Here, a biotinylated primer isused during PCR amplification, which pro-vides a substrate for binding of the fluores-cent streptavidin-R-phycoerythrin that resultinto labeling of hybridized beads (Armstronget al. 2000; Spiro et al. 2000). High
fluorescence is generated due to binding ofamplicon with the microspheres.
2. Competitive DNA hybridization: In thismethod, unlabeled PCR amplicons are usedfor hybridization in combination with bio-tinylated competitor oligonucleotides. Inabsence of target DNA competitor DNAbinds to the microsphere and produce highfluorescence and when target DNA is presentit binds to the competitor DNA which in turnis not able to bind to the microsphere and lowfluorescence is generated, which is contrast-ing to the direct hybridization method.
3. Solution-based chemistries with micro-sphere capture: These chemistries exploitthe natural properties of DNA polymerasesand ligases and incorporate a capturesequence during the solution-based reactions.In these techniques, universal microsphereswith nonspecific capture sequences are used.These universal capture sequences mainlyinclude ZipCode/cZipCode, xTAG of TmBiosciences, Luminex Molecular Diagnos-tics, Inc., Toronto, Canada and EraCode ofEraGen, Madison, WI (Johnson et al. 2004).ZipCode/cZipCode were the first universalcapture sequences those were used to detectSNP’s using SBCE (Chen et al. 2000;Iannone et al. 2000; Taylor et al. 2001; Yeet al. 2001). ZipCode sequences are basically25 bp long random sequences of Mycobac-terium tuberculosis genome (Chen et al.2000). They are used by adding a uniqueZipCode to the 50 end of the capture probeand tagging of microspheres with its com-plementary sequence (cZipCode). In xTAG,only three nucleotides out of four are used toavoid nonspecific hybridization with thenaturally occurring sequences. Further, theyare thermodynamically matched to avoidvariability in their hybridization efficiency.This technology is used in commercial assaysconducted by Luminex. In EraCode, synthe-sized isoguanosine and 5-Me-isocytosinebases are used that are based on the expandedgenetic alphabet of MultiCode technology.The isoguanosine and 5-Me-isocytosinebases pairs with each other but they do not
6 DNA Microarray 79
pair with natural bases due to which EraCodesequences are highly specific. Solution-basedchemistries coupled with subsequent micro-sphere capture include following procedures:
(a) Allele-specific primer extension (ASPE)or target-specific primer extension(TSPE): In ASPE or TSPE a capture primerwith a unique 50 sequence followed by atarget-specific sequence is used. This primeris extended by DNA polymerase if targetDNA is present to provide the complemen-tary base for the nucleotide present on 30
end. Biotinylated deoxynucleotide triphos-phate is used in ASPE and/or TSPE toachieve labeling.
(b) Oligonucleotide ligation assays (OLA):Same as in ASPE and/or TSPE a captureprimer with a unique 50 sequence followedby a target-specific sequence is used inOLA. The difference is in the enzymaticreaction which is ligase dependent in OLA.In OLA a biotinylated probe homologous totarget DNA is used in addition to the captureprimer. Here, if target DNA is present in thesample, capture primer and reporter probeligates.
(c) Single-base-chain extension (SBCE): Thisprocedure is used for detection of multipleSNP in one run. Here, independent reactionsare required for each nucleotide query and
1. Microspheres 5.6 µm in diameter are filled with different relative concentrations of an
infrared dye and a red dye to create 100 beads, each with a unique spectral identity
2. Potential targets are amplified using a biotinylated primer and then denatured and hybridized to microspheres tagged
with target-specific sequence probes
3. Probe-target hybridization is measured using a streptavidin-bound green
fluorescence
4. Flow cytometry is used to analyze the microsphere
suspension
5. A red laser is used to determine the spectral identity of the bead and, therefore, the
probe being analyzed
6. The reporter fluorochrome is excited by a green laser, which
quantifies the probe-target reaction on the microsphere
surface
Fig. 6.6 Steps involved in microarray experiment using suspension bead array
80 A. M. Charpe
for identifying a single SNP one probe witha unique capture sequence is used to identifythe possible alleles in separate wells havinga different dideoxynucleoside triphosphatein each well. Likewise, in the well in whichhomologous capture and target sequence ispresent, a biotinylated dideoxynucleosidetriphosphate gets incorporated, hence thefurther extension gets terminated.
Despite the low feature density due to theavailability of universal capture sequences, highability of multiplexing, relative simplicity, andaffordability suspension bead arrays are highlysuitable for high-throughput nucleic acid detec-tions including clinical diagnostics if due care istaken to avoid post-amplification intra- andinter-run contaminations.
6.4.3 Electronic Microarrays
Electronic microarray technology is introducedby Nanogen, San Diego, CA. In contrast to allother microarrays that depend on passivemovement of complimentary sequences forhybridization, in electronic microarrays theirmovement is directed by applying electric field.For this purpose in microelectronic cartridges,complementary metal oxide semiconductortechnology is used, e.g., NanoChip 400 of Na-nogen (Sosnowski et al. 1997). Each NanoChipcartridge is provided with 12 connectors that cancontrol 400 individual test sites. By applicationof positive current to one or more test sites onthe microarray, nucleic acids which are basicallynegatively charged move towards specific sites,or features. When electronically directed bio-tinylated probes reaches to the target site itforms streptavidin-biotin bond with the strepta-vidin present on the microarray surface. Oncethe probes reaches to their target, positive cur-rent is removed from the active features and newtest sites are activated. After the probes areattached to the features, fluorescently labeled
target DNA is applied to the microarray. TargetDNA can hybridize with the probe either pas-sively or by concentrating them electronically.The most common format is the application ofprobe first, although amplicon may also beapplied first and sometimes sandwich assays arealso used. Irrespective of the format used, ifhybridization takes place, presence of fluores-cent reporters can be detected by scanning whichis then analyzed (Fig. 6.7) (Miller and Tang2009).
In electronic microarrays multiplex detectioncan be achieved at an individual test site becausemultiple probes, each with a distinct fluoro-phore, can be sequentially directed to the samefeature, e.g., Nanogen’s RVA ASR in which P1,parainfluenza virus type 1; P2, parainfluenzavirus type 2; P3, parainfluenza virus type 3; FB,influenza B virus; FA, influenza A virus; RSV,respiratory syncytial virus; BKGD, backgroundcan be detected simultaneously. On thesemicroarrays single or multiple samples can beused for hybridization to multiple test sites.Further, the commercially available electroniccartridge NanoChip is a universal blank chip,hence the features of the microarray can bedirectly specified by the user and therefore thesemicroarrays are highly flexible and cost-effective.At present, electronic microarrays facilitate adensity of only 400 spots which is sufficient formost of the clinical diagnostic assays.
6.5 Design of DNA Microarrays
Depending on the number of probes underexamination, costs, customization requirements,and the type of scientific question to beanswered microarrays can be manufactured indifferent ways. Arrays may have as few as 10probes or up to 2.1 million lm-scale probes. Thedesigning of microarrays depends on two chan-nels (two color microarrays) or one channel ofdetection (one color microarray).
6 DNA Microarray 81
6.5.1 Two-Color Microarrays or Two-Channel Microarrays
Two-color microarrays or two-channels aretypically hybridized with cDNA prepared fromtwo samples to be compared (e.g., diseased tis-sue versus healthy tissue) and that are labeledwith two different fluorophores (Shalon et al.1996; Fig. 6.8). Fluorescent dyes commonlyused for cDNA labeling include Cy3, which hasa fluorescence emission wavelength of 570 nm(corresponding to the green part of the lightspectrum), and Cy5 with a fluorescence emissionwavelength of 670 nm (corresponding to the redpart of the light spectrum). The two Cy-labeledcDNA samples are mixed and hybridized to asingle microarray that is then scanned in amicroarray scanner to visualize fluorescence ofthe two fluorophores after excitation with a laser
beam of a defined wavelength. Relative inten-sities of each fluorophore may then be used inratio-based analysis to identify up-regulated anddown-regulated genes (Tang et al. 2007a). Insitu synthesised oligonucleotide microarraysoften carry control probes designed to hybridizewith RNA spike-ins. The degree of hybridizationbetween the spike-ins and the control probes isused to normalize the hybridization measure-ments for the target probes. Although absolutelevels of gene expression may be determined inthe two-color array in rare instances, the relativedifferences in expression among different spotswithin a sample and between samples is thepreferred method of data analysis for the two-color system. Examples: Dual-Mode platform ofAgilent, DualChip platform for colorimetricSilverquant labeling of Eppendorf, and Arrayitof TeleChem International.
1. A positive electric current is applied to test sites, facilitating the active movement and concentration of negatively charged DNA
probes to the activated locations
2. Once the first probe is bound to its targeted location(s) by streptavidin-biotin bonds, the test site(s) can be deactivated, and current
can be applied to a different test site
3. Upon application of the probes to targeted test sites, extracted and amplified nucleic
acids from a sample passively hybridize to the microarray surface
4. If hybridization occurs, secondary probes that are specific for the target
and that contain a nonspecific detector sequence binds
5. Secondary fluorescent detector oligonucleotides are used to measure
positive hybridization reactions
This process is repeated until all the probes are arrayed
Fig. 6.7 Steps involved in detection using electronic microarray
82 A. M. Charpe
6.5.2 Single-Channel Microarraysor One-Color Microarrays
In single-channel microarrays or one-colormicroarrays, the arrays provide intensity data foreach probe or probe set indicating a relative levelof hybridization with the labeled target. However,they do not truly indicate abundance levels of agene but rather relative abundance when com-pared to other samples or conditions when pro-cessed in the same experiment. Each RNAmolecule encounters protocol and batch-specificbias during amplification, labeling, and hybrid-ization phases of the experiment making com-parisons between genes for the same microarrayuninformative. The comparison of two conditionsfor the same gene requires two separate single-dye hybridizations. Examples are AffymetrixGeneChip, Illumina BeadChip, Agilent single-channel arrays, the Applied Microarrays Code-Link arrays, and the Eppendorf DualChip and
Silverquant arrays. One strength of the single-dyesystem is that an aberrant sample cannot affect theraw data derived from other samples, becauseeach array chip is exposed to only one sample (asopposed to a two-color system in which a singlelow-quality sample may drastically impinge onoverall data precision even if the other sample wasof high quality). Another benefit is that data aremore easily compared to arrays from differentexperiments so long as batch effects have beenaccounted for. A drawback to the one-color sys-tem is that, when compared to the two-color sys-tem, double the number of microarrays arerequired to compare samples within anexperiment.
6.6 Basic Applications of DNAMicroarrays
6.6.1 Gene Expression Profiling
DNA microarrays can be used to detect DNA (asin comparative genomic hybridization), or detectRNA (most commonly as cDNA) that may ormay not be translated into proteins. The processof measuring gene expression via cDNA is calledexpression analysis or expression profiling. In anmRNA or gene expression profiling experiment,the expression levels of thousands of genes aresimultaneously monitored to study the effects oftreatments, diseases, and developmental stageson gene expression. For example, microarray-based gene expression profiling can be used toidentify genes whose expression is changed inresponse to pathogens or other stresses as com-pared to healthy tissues (Adomas et al. 2008).
6.6.2 Comparative GenomicHybridization
This is meant for assessing genome content indifferent cells or closely related organisms, forexample, tumerogenic cells (Moran et al. 2004).
Fig. 6.8 A typical two-color microarray experiment(Source http://en.wikipedia.org/wiki/DNA_microarray)
6 DNA Microarray 83
6.6.3 GeneID
These are small microarrays to check IDs oforganisms in food and feed (Mascos andSouthern 1992), mostly combining PCR andmicroarray technology. For example, geneticallymodified organisms, mycoplasmas in cell cultureand pathogens for disease detection.
6.6.4 Chromatin Immunoprecipitationon Chip
DNA sequences bound to a particular protein canbe isolated by immunoprecipitating that protein(ChIP), these fragments can be then hybridizedto a microarray (such as a tiling array) todetermine the protein binding site occupancythroughout the genome. For example, protein toimmunoprecipitate are histone modifications(H3K27me3, H3K4me2, H3K9me3, etc.), Poly-comb-group protein (PRC2:Suz12, PRC1:YY1)and trithorax-group protein (Ash1) to study theepigenetic landscape and RNA Polymerase II tostudy the transcription landscape.
6.6.5 DamID
Analogously to ChIP, genomic regions bound bya protein of interest can be isolated and used toprobe a microarray to determine binding siteoccupancy. Unlike ChIP, DamID does not requireantibodies but makes use of adenine methylationnear the protein’s binding sites to selectivelyamplify those regions, introduced by expressingminute amounts of protein of interest fused tobacterial DNA adenine methyltransferase.
6.6.6 SNP Detection
SNP detection is for identifying single nucleo-tide polymorphism among alleles within orbetween populations (Hacia et al. 1999). Several
applications of microarrays make use of SNPdetection, e.g., genotyping, forensic analysis,measuring predisposition to disease, identifyingdrug-candidates, evaluating germline mutationsin individuals, somatic mutations in cancers,assessing loss of heterozygosity, and geneticlinkage analysis.
6.6.7 Alternative Splicing Detection
An exon junction array design uses probesspecific to the expected or potential splice sitesof predicted exons for a gene. It is of interme-diate density or coverage, to a typical geneexpression array (with 1–3 probes per gene) anda genomic tiling array (with hundreds or thou-sands of probes per gene). It is used to assay theexpression of alternative splice forms of a gene.Exon arrays have a different design; employingprobes designed to detect each individual exonfor known or predicted genes, and can be usedfor detecting different splicing isoforms.
6.6.8 Fusion Genes Microarray
A fusion gene microarray can detect fusiontranscripts, e.g., from cancer specimens. Theprinciple behind this is building on the alterna-tive splicing microarrays. The oligo designstrategy enables combined measurements ofchimeric transcript junctions with exon-wisemeasurements of individual fusion partners.
6.6.9 Tiling Array
Genome tiling arrays consist of overlappingprobes designed to densely represent a genomicregion of interest, sometimes as large as anentire human chromosome. The purpose is toempirically detect expression of transcripts oralternatively splice forms, which may not havebeen previously known or predicted.
84 A. M. Charpe
6.7 Specialized Applicationsof DNA Microarrays
DNA microarrays have various specific applica-tions to Cancer biology, Microbiology, Plantscience, Medicine, Environmental Science,Nephrology, etc. Few of them are explained here.
6.7.1 Applications to Cancer Biology
Cancer is caused by the accumulation of geneticand epigenetic changes resulting from thealtered sequence or expression of cancer-relatedgenes, such as oncogenes or tumor suppressorgenes, as well as genes involved in cell cyclecontrol, apoptosis, adhesion, DNA repair, andangiogenesis. Because gene expression profilesprovide a snapshot of cell functions and pro-cesses at the time of sample preparation, com-prehensive combinatorial analysis of the geneexpression patterns of thousands of genes intumor cells and comparison to the expressionprofile obtained with healthy cells should pro-vide insights concerning consistent changes ingene expression that are associated with tumorcellular dysfunction and any concomitant regu-latory pathways. Microarray technology hasbeen widely used in the past 10 years to inves-tigate tumor classification, cancer progression,and chemotherapy resistance and sensitivity(Macgregor and Squire 2002). Although themajor limiting factors for routine use in a clin-ical setting at present are cost and access to themicroarray technology, it is likely that costs willdecrease in the near future and that the tech-nology will become increasingly user friendlyand automated. In this section, few examples arediscussed to demonstrate that expression arrayscan be used to gain a better understanding of thebasic biology, diagnosis and treatment of cancer.
6.7.1.1 Identifying Single NucleotidePolymorphism (SNP)
SNP’s are common DNA sequence variationshaving great significance for biomedical
research. In human genome at least one out ofevery thousand nucleotide is observed to havepolymorphism. Most of the SNP’s are harmlessbut few of them that are located in codingregions of the gene alter the composition ofamino acids, thus the functions of encodedproteins. Similarly, SNP’s located in the non-coding regions are also important if they affectthe splicing of the gene, or alter the promoter,enhancer, silencer or any recognition site forDNA binding proteins (Risch 2000). For exam-ple, a SNP in 50 untranslated region of theRAD51 gene have been identified that may beassociated with an increased risk of breast can-cer and a lower risk of ovarian cancer amongBRCA2 mutation carriers (Wang et al. 2001).
6.7.1.2 Detection of MutationsHigh throughput methods like microarray assaysare found useful in rapid detection of mutationsamong large genes like BRCA1, BRCA2. Forexample, a high density oligonucleotide arraywith about 96,000 oligonucleotides of 20 bp sizehave been developed by Hacia et al. (1996) thatcould detect a wide range of heterozygousmutations in the 3.45 Kb exon 11 of the BRCA1gene. Wen et al. (2000) found that oligonu-cleotide based detections are more accurate thanDNA sequencing in identifying p53 mutations inovarian tumors.
6.7.1.3 Molecular Classificationof Tumors
Improvements in tumor classification are centralto the development of novel and individualizedtherapeutic approaches. Histologically indistin-guishable tumors often show significant differ-ences in clinical behavior, and sub classificationof these tumors based on their molecular profilesmay help explain why these tumors respond sodifferently to treatment (Macgregor and Squire2002).
For example, (1) microarray technology wasapplied to develop innovative classifications ofleukemias using microarray analysis based on
6 DNA Microarray 85
neighborhood analysis and the utilization oftumor class predictors. This strategy was able todistinguish between acute myeloid leukemia andacute lymphocytic leukemia without supervisoryanalysis (Golub et al. 1999); (2) gene expressionpattern analysis has been used to classify, at themolecular level, breast tumors (Sorlie et al.2001), B cell lymphoma (Alizadeh et al. 2000),cutaneous melanoma (Bittner et al. 2000), andlung adenocarcinoma (Garber et al. 2001;Bhattacharjee et al. 2001); (3) by analyzingmolecular profiles of 50 nonneoplastic andneoplastic prostate samples, signature expres-sion profiles of healthy prostate, benign prostaticneoplasia, localized prostate cancer, and meta-static prostate cancer has been established(Dhanasekaran et al. 2001). These studiesestablished the feasibility of combining large-scale molecular analysis of expression profileswith classic morphologic and clinical methodsof staging and grading cancer for better diag-nosis and outcome prediction.
6.7.1.4 Discoveries of Novel GenesMany tumor suppressor genes and oncogenes areregulated by the expression of other genes attranscription level. Even some of them like p53and Myc are transcription factors. Upon activa-tion, p53 induces growth arrest or apoptosis bytranscriptionally activating its target genes. Withthe help of microarray-based expression profilingtarget genes for several gene products have beenidentified which directly or indirectly regulatestranscription. For example, oligonucleotide arrayhaving 6,000 genes is used to identify p53 targetgenes (Zhao et al. 2000). Similarly, microarray-based expression profiling have been used toidentify Gadd45 as one of the targets of BRCA1tumor suppressor gene (Harkin et al. 1999).
6.7.1.5 Detection of Sensitivityto Drugs
Despite considerable advances in cancer treat-ment, acquired resistance to chemotherapeuticdrugs continues to be a major obstacle in patient
treatment and overall outcome. Anticancer drugresistance is thought to occur through numerousmechanisms, and microarrays offer a newapproach to studying the cellular pathwaysimplicated in these mechanisms and in predict-ing drug sensitivity and unexpected side effects.Most array studies have been carried out usingcancer cell lines that are rendered resistant tocommonly used anticancer drugs. Obtainingfurther insights into the mechanism of action ofanticancer drugs and the diverse pathwaysinvolved in drug resistance may eventually beinvaluable for design of more strategic treat-ments that are most appropriate for an individualtumor (Macgregor and Squire 2002).
For example, (1) in an attempt to obtainmolecular fingerprinting of anticancer drugs incancer cells the expression profiles of doxoru-bicin-induced and -resistant cancer cells weremonitored (Kudoh et al. 2000); (2) similarly, inanother study, a subset of 1,400 genes wereanalyzed to study the correlation betweenexpression profiles and drug mechanism ofaction of a panel of 118 anticancer drugs (Rosset al. 2000; Scherf et al. 2000).
6.7.1.6 Tumor Metastasis RelatedStudies
Conversion of noninvasive tumors into invasivetumors is known as metastasis when cancer cellsstart spreading from one organ to another organor tissue with blood stream or lymphatic system.DNA microarrays have been used to identify thegenes involved in metastasis.
For example, clonal relationship of 22 livertumor foci from six patients have been investi-gated by Cheung et al. (2002) with the help ofcDNA microarray having 23,000 genes. With anaim to identify metastasis-related genes, theycould identify 63 up-regulated genes, of which39 were known genes and 24 were expressedsequence tags and 27 down regulated genes, ofwhich 14 were known genes and 13 wereexpressed sequence tags. Similar studies havebeen conducted with osteosarcoma, colorectaltumor and brain metastasis.
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6.7.1.7 Identification of MolecularMarkers Specific to Tumor
Several research groups have focused on iden-tifying subsets of genes that show differentialexpression between healthy tissues or cell linesand their tumor counterparts to identify bio-markers for several solid tumors, includingovarian carcinomas, oral cancer, melanoma,colorectal cancer, and prostate cancer.
In a recent study, a subset of genes showingdifferential expression between healthy ovariesand ovarian tumors has been reported. Some ofthese genes, such as metallothionein 1G, whichwas found to be up-regulated in tumor samples,are implicated in resistance to the anticancerdrug cisplatin and might be an indicator ofpretreatment resistance of these tumors to cis-platin (Bayani et al. 2002). Other gene identifiedin this study is the osteopontin gene, which wasstrongly up-regulated in some tumor samplesthat have been shown to be secreted in the serumof patients with metastatic cancer, might be anexcellent candidate for biomarkers of tumorprogression in EOC (Singhal et al. 1997).
One of the most important challenges investi-gators are facing while using microarray analysisis determining which of the plethora of new dif-ferentially expressed genes is biologically rele-vant to the tumor system being studied. Evenwhen rigorous efforts are made to minimize thenumber of variables in a microarray study, theremay be an unmanageable number of differentiallyexpressed genes that will contribute excessivebackground values. Therefore, combiningexpression microarray analysis with otherapproaches, particularly cytogenetics techniques,such as spectral karyotyping and comparativegenomic hybridization array (CGH) (Pollack et al.1999), offers the possibility to focus on signifi-cantly smaller subsets of genes of direct relevanceto tumor biology (Bayani et al. 2002). A combi-nation of expression arrays and CGH array tech-niques was used on breast cancer cell lines and hasidentified a limited number of genes that are bothamplified and over expressed (Monni et al. 2001).
Finally, validation of the relative expressionobtained from genome-wide microarray analysis
is critical. Several approaches can be chosenfrom basic Northern analysis or semiquantitativereverse transcription-PCR to in situ hybridiza-tion (ISH) using tissue microarrays. For exam-ple, expression of several candidate genesassociated with prostate cancer has been ana-lyzed (Mousses et al. 2002) those were previ-ously identified by cDNA microarray analysis.Tissue microarrays constructed from 544 histo-logical biopsies were analyzed by ISH usingRNA probes and/or by immunohistochemistry(IHC) using antibodies. There was excellentcorrelation between the cDNA microarrayresults and the results obtained with ISH andNorthern blot analysis. In addition, proteinexpression assessed by IHC was also consistentwith RNA expression. Similarly, comparabletechnologies to confirm over expression ofhepsin and PIM-1 in prostate cancer have beenused (Dhanasekaran et al. 2001).
6.7.2 Applications to Microbiology
DNA microarrays can be applied to various aspectsof microbial studies like microbial detection andidentification, determination of antimicrobial drug,drug resistance, microbial typing, microbial geneexpression profiling, host gene expression profilingduring microbial infections, and determination ofhost genomic polymorphism.
6.7.2.1 Detection and Identificationof Microbes
DNA microarrays could potentially be an assaymethod to address multiple questions for speciesidentification in both clinical and environmentalsettings. It identifies their phylogenetic statusbased on unique 16S rRNA sequences and pro-vides information related to the presence ofantibiotic markers and pathogenicity regions (Yeet al. 2001).
For example, (1) a DNA probe array have beendescribed for species identification and detectionof rifampin resistance in M. tuberculosis (Troeschet al. 1999). In this 70 mycobacterial isolates from
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27 different species and 15 rifampin-resistantstrains were tested. A total of 26 of the 27 speciesas well as all of the rifampin-resistant mutantswere correctly identified. (2) For field applica-tions, a portable system for microbial samplepreparation and oligonucleotide microarrayanalysis has also been reported (Bavykin et al.2001). This portable system contained threecomponents: (a) a universal silica mini-columnfor successive DNA and RNA isolation, frac-tionation, fragmentation, fluorescent labeling,and removal of excess free label and short oligo-nucleotides; (b) microarrays of immobilized oli-gonucleotide probes for 16S rRNA identification;and (c) a portable battery-powered device forimaging the hybridization offluorescently labeledRNA fragments on the arrays. Beginning withwhole cells, it takes approximately 25 min toobtain labeled DNA and RNA samples and anadditional 25 min to hybridize and acquire themicroarray image using a stationary image anal-ysis system or the portable imager. (3) Respira-tory viral pathogen has been detected inconnection with multiplex PCR amplification. (4)Simultaneous detection and typing ofhuman papillomaviruses and (5) rapid detectionand characterization of methicillin resistantStaphylococcus aureus, have also become possi-ble through DNA microarrays.
6.7.2.2 Virulence Factor Determinationof Pathogenic Microbes
Many genes associated with virulence are reg-ulated by specific conditions. One way todetermine the candidate virulence factors is toinvestigate the genome-wide gene expressionprofiles under relevant conditions, such asphysiological changes during interaction withthe host. A second approach would rely oncomparative genomics (Ye et al. 2001).
For example, in a genome comparison studyamong Helicobacter pylori strains, a class ofcandidate virulence genes was identified by theircoinheritance with a pathogenicity island usingDNA microarray technique. The whole genomemicroarray of H. pylori was also shown to be aneffective method to identify differences in gene
content between two H. pylori strains that inducedistinct pathological outcomes. It is demon-strated that the ability of H. pylori to regulateepithelial cell responses related to inflammationdepends on the presence of an intact cag patho-genicity island (Salama et al. 2000).
6.7.2.3 Responses of Hostto Pathogenic or ResidentMicrobes
DNA microarray is useful in study of the hosttranscriptional profiles and identifying the dif-ferentially expressed genes in host-microbeinteraction. Such experiments demonstrate thathost genomic transcriptional profiling, in com-bination with functional assays to evaluate sub-sequent biological events, provides insight intothe complex interaction of host and humanpathogens. At the same time, such studies helpin developing our understanding about theessential nature of the interactions between res-ident microorganisms and their hosts.
For example, (1) the host transcriptional pro-files during the interaction of Bordetella pertus-sis with a human bronchial epithelial cell lineBEAS-2B were investigated using high-densityDNA microarrays (Belcher et al. 2000). Theearly transcriptional response to this pathogen isdominated by the altered expression of cyto-kines, DNA-binding proteins, and NFkB-regu-lated genes. It was found that B. pertussis inducesmucin gene transcription by BEAS-2B cells andthen counters this defense by using mucin as abinding substrate. This result indicates the hostdefensive and pathogen counter-defensive strat-egies. (2) A DNA microarray was also used toidentify the host genes that were differentiallyexpressed upon infection by Psuedomonasaeruginosa to the A549 lung pneumocyte cellline (Ichikawa et al. 2000). Differential expres-sion of genes involved in various cellular func-tions was found, and one of those genes encodesthe transcription factor interferon regulatoryfactor-1. (3) Further, monitoring of cellularresponses to Listeria monocytogenes and Chla-mydia pneumoniae has also been reported(Cohen et al. 2000; Coombes and Mahony 2001).
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(4) DNA microarrays have been used to inves-tigate the global intestinal transcriptionalresponses to the residential colonization of Bac-teroides thetaiotaomicron, a prominent compo-nent of the normal mouse and human intestinalmicroflora (Hooper et al. 2001). The resultsreveal that this commensal bacterium modulatesthe expression of genes involved in severalimportant intestinal functions, including nutrientabsorption, mucosal barrier fortification, xeno-biotic metabolism, angiogenesis, and postnatalintestinal maturation.
6.7.2.4 Study of Mode of Actionof Drugs, Inhibitors and ToxicCompounds
Inhibition of a particular cellular process mayresult in a regulatory feedback mechanism,leading to changes in gene expression patterns.Exploring the gene expression profiles with DNAmicroarrays may reveal information on the modeof action for drugs and inhibitors or toxic com-pounds. Insights gained from this approach maydefine new drug targets and suggest new methodsfor identifying compounds that inhibit thosetargets. In addition to the alternation in geneexpression patterns related to the drug’s mode ofaction, drugs can induce changes in genes relatedto stress responses that are linked to the toxicconsequences of the drug. The secondary effectsof a drug may reveal information on the potentialresistance mechanism, which may help to designthe drugs that have fewer side effects but havehigh efficacy by reducing the ability of bacteriato neutralize the drug. Each type of compoundoften generates a signature pattern of geneexpression. A database populated with thesesignature profiles can serve as a guide to eluci-date the potential mode of action as well as sideeffects of uncharacterized compounds.
For example, DNA microarray hybridizationexperiments have been conducted in M. tuber-culosis to explore the changes in gene expressioninduced by the antituberculous drug isoniazid(Wilson et al. 1999). Isoniazid selectively
interrupts the synthesis of mycolic acids, whichare branched b-hydroxy fatty acids. Microarrayexperiments showed that isoniazid induced sev-eral genes that encode proteins that are physio-logically relevant to the drug’s mode of action,including an operonic cluster of five genesencoding type II fatty acid synthase enzymes andfbpC, which encodes trehalose dimycolyltransferase.
6.7.2.5 Studies Related to Evolutionand Epidemiology of Microbes
DNA microarrays can be used to explore thevariability in genetic content and in geneexpression profiles within a natural populationof the same or related species and between theancestor and the descendents. As a result, itprovides very rich information on the molecularbasis of microbial diversity, evolution, and epi-demiology (Ye et al. 2001).
For example, genomes within the species ofM. tuberculosis have been compared with a highdensity oligonucleotide microarray to detectsmall-scale genomic deletions among 19 clini-cally and epidemiologically well-characterizedisolates (Kato-Maeda et al. 2001). This studyreveals that deletions are likely to containancestral genes whose functions are no longeressential for the survival of organism, whereasgenes that are never deleted constitute the min-imal mycobacterial genome. As the amount ofgenomic deletion increased, the likelihood thatthe bacteria will cause pulmonary cavitationdecreased, suggesting that the accumulation ofmutations tends to diminish their pathogenicity.
6.7.2.6 Process Optimizationand Pathway Engineering
Traditional approaches of biocatalysis optimi-zation use random screening, mutagenesis, andengineering improvement. While these methodsare still very effective, a better understanding ofthe underlying physiology using genomic tools
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can accelerate these efforts. Informationobtained by the DNA microarrays can helppathway engineering and process optimizationin following ways.1. Regulatory circuitry and coordination of gene
expression among different pathways underdifferent growth conditions can be measuredby DNA microarray
2. The physiological state of the cells duringfermentation can be assessed by the genome-wide transcriptional patterns
3. DNA microarray can help in identifyinggenes involved in a production process if theyare coregulated
4. The differences in genetic contents andexpression profiles between wild-type andimproved strains can be compared
5. The actual array data can be incorporated intothe mathematical models to describe a cel-lular process
6. General applications of DNA microarraytechnology to understand microbial physiol-ogy will continue to generate very largeamounts of information that will ultimatelybenefit the pathway engineering and fer-mentation optimization effort.Current research in using array information in
pathway engineering and bioprocessing is at itsearly stage. Arrays containing genes involvedcentral metabolism, key biosyntheses, someregulatory functions, and stress response havebeen used to investigate the metabolic responsesto protein overproduction and metabolic fluxes inE. coli (Oh and Liao 2000a, b; Gill et al. 2001).Gene array analysis was also used as a tool toinvestigate the differences in the expressionlevels for 30 genes involved in xylose catabolismin the parent, strain B, and the engineered strain,KO11 (Tao et al. 2001). Increased expression ofgenes involved in xylose catabolism is proposedas the basis for the increase in growth rate andglycolytic flux in ethanologenic KO11 strain.Refer Table 6.1 for information on differenttypes of micro arrays used in microbial systemsand useful links to various websites with infor-mation on microbial genome.
6.7.3 Applications to Plant Science
DNA microarrays are playing significant role inunderstanding the expressions of genes in situa-tions like effect of biotic stresses like disease,insect and herbivore attacks; effect of abioticstresses like cold, heat, soil salinity, water defi-ciency, etc.; effect of circadian rhythm on diur-nal cycle; and various physiological functionsand genomic composition of plants. Some of theapplications are explained here.
6.7.3.1 Studies on Biotic StressesIn plants, biotic stresses are responsible forreducing the yield potential. Studies using DNAmicroarrays has provided some understanding ofdifferential gene expressions and defenseresponses to biotic stresses like diseases, insectpests, and herbivore attack on plants. Thegenomic expressions in biotic stresses could beunderstood largely due to the applications ofcDNA microarrays (Ahroni and Vorst 2001).Some of them are given below based on thework mostly done on Arabidopsis.
For example, (1) the expression of two Ara-bidopsis accessions having rosette leaves hasbeen studied with the help of cDNA microarrayhaving 673 clones (Kehoe et al. 1999); (2) the traitfor defense response to pathogen in Arabidopsishas been mapped by oligonucleotide microarrayhaving 412 polymorphisms (Cho et al. 1999);(3) cDNA microarrays having 150 clones havebeen used to study the response of Arabidopsisto mechanical wounding and insect feeding(Reymond et al. 2000); (4) cDNA microarrayhaving 2375 clones has been employed tounderstand the response to treatments withdefense-related signaling molecules and fungalpathogen in Arabidopsis (Schenk et al. 2000);(5) in Arabidopsis genomic expressions associ-ated with systemic acquired resistance (SAR)have been studied by using cDNA microarrayhaving 10,000 clones (Maleck et al. 2000); (6)expression of downstream genes in MAP kinase4 signaling pathways has also been identified
90 A. M. Charpe
Table 6.1 Links of sources with information on microbial genomes and DNA microarray construction and appli-cations (Ye et al. 2001)
S.N. Web link Reference and availableinformation
1 http://www.affymetrix.com/ It provides commercial high-density oligonucleotide arrays andservices
2 http://www.operon.com/ Operon. It provides 70 meroligonucleotide array sets andprimer synthesis service
3 http://www.genosys.com/ SigmaGenosys. It providesmembrane arrays and wholegenome array sets. Other servicesinclude primer synthesis
4 http://www.eurogentec.com/ Eurogentec. It providesoligonucleotides and wholegenome microbial arrays. It alsoperforms custom arrayexperiments
5 http://www.nanogen.com/ Nanogen. Microelectronics10–400 bp fragments
6 http://www.cmgm.stanford.edu/pbrown/ The Brown laboratory home page.It includes a complete guide tomicroarraying for the molecularbiologist and other links
7 http://www.jgi.doe.gov/tempweb/JGI_microbial/html Joint Genome Institute. This sitecontains information on thecurrent microbial genome-sequencing project
8 http://www.tigr.org/tigr-scripts/CMR2/CMR HomePage.spl Tigr’s. Comprehensive MicrobialResource (CMR) home page
9 http://www.genome.wi.mit.edu/genome_software/other/primer3.html Software Primer3 for primerdesign
10 http://www.microarrays.org/index.html Protocols and other information
11 http://www.arrayit.com/index.html TeleChem International. Itprovides slides, software and otherservices
12 http://www.corning.com/ Corning. It provides slides andcommercial DNA arrays
13 http://www.genemachines.com/ GeneMachines. It provides spotterand other robotics
14 http://www.axon.com/ Axon Instruments. Scanner
15 http://www.mdyn.com/ Molecular Dynamics. It providesspotter and scanners
16 http://www.stat.berkeley.edu/users/ Terry Speed’s microarray dataanalysis group page
17 http://www.rana.lbl.gov/EisenResearch.html Michael Eisen’s software Clusterand TreeView
18 http://www.lionbioscience.com/ Lion Bioscience. It providesarraySCOUT software and anintegrated database platformbased on SRS system
(continued)
6 DNA Microarray 91
with the help of cDNA microarray having 9,861clones (Peterson et al. 2000); (7) cDNA micro-array having 11,521 clones have been used tosolve large-set of biological questions, e.g.,amino acid metabolism, apoptosis, development,effect of environmental conditions, effect ofhormonal treatment, effect of metals, functionsof mitochondria, attack by pathogen, stress,RNA stability, comparative genomics and viru-ses in Arabidopsis (Schaffer et al. 2000; Wismanand Ohlroggge 2000); and (8) in lima beanresponses to herbivory and herbivore-inducedvolatiles have been studied with the help ofcDNA microarray having 2,032 clones.
6.7.3.2 Studies on Abiotic StressesLike biotic stresses abiotic stresses also playsignificant role in healthy growth of plants.Abiotic stresses like unfavorable temperature,humidity, light, soil pH, soil water, etc.,adversely affect plant physiology, therebyaffecting its proper growth and yield potential.Therefore, understanding of biochemical chan-ges taking place in plants during stressed con-dition may help in minimizing the adverse effecton yield by adopting appropriate management
practices or by developing tolerant transgenicvarieties (Ahroni and Vorst 2001). Most of suchstudies have been conducted using cDNA, DNAand oligonucleotide microarrays.
For example, (1) Genomic expressions ofArabidopsis under drought and cold stresses areexplained with cDNA microarray having 1,300clones (Seki et al. 2001). (2) Response of Syn-echocystis sp to high light has been studied withthe help of DNA microarray having 1.0 kb PCRfragments of 3079 PCC6803 clones (Hiharaet al. 2001). (3) Similarly, expression of cold-regulated genes in hik33 mutant of Synecho-cystis sp have been understood with the use ofDNA microarray prepared with 1.0 kb PCRfragments of 3079 PCC6803 clones (Suzukiet al. 2001). (4) Gene expressions due to saltstress have been studied in rice with the help ofcDNA microarray having 1,728 clones (Kawasakiet al. 2001). (v) At Stanford university geneexpression following exposure to high salinity inice plant and Arabidopsis have been studied byArabidopsis Functional Genomics Consortiumusing DNA, cDNA and oligonucleotide arrayshaving 2,600 clones (Bohnert et al. 2001,http://afgc.stanford.edu).
Table 6.1 (continued)
S.N. Web link Reference and availableinformation
19 http://www.sigenetics.com/cgi/SiG.cgi/index.smf Silicon Genetics. It providesGeneSpring Suite for dataanalysis, presentation and storage
20 http://www.genome-www4.stanford.edu/MicroArray/SMD/ Stanford Microarray Database
21 http://www.genomics.lbl.gov/*ecoreg/ The E. coli regulation consortium.A site for primary and secondarydata on pathway regulation
22 http://www.sghms.ac.uk/depts/medmicro/bugs/ Bacterial microarrays at St.George’s Hospital Medical School
23 http://www.web.wi.mit.edu/young/location/ Methods for genome-wideprotein–DNA binding studies
24 [email protected] PrimeArray software for genomescale primer design
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6.7.3.3 Studies on Diurnal ChangesDiurnal changes regulated by circadian clock arewell understood in human beings. Betterunderstanding of circadian rhythm in plants mayhelp in selecting area specific crop, responsivevariety and to device efficient crop managementpractices for sustainable food security. Effortshave been made to understand the existence ofsuch circadian rhythm in plants by using oligo-nucleotide microarray representing 8,200 genes(Harmer et al. 2000) and cDNA microarrayhaving 11,521 clones (Schaffer et al. 2001).
6.7.3.4 Studies on Physiology of PlantsUnderstanding biochemical details of normalphysiology of plants may help in improving themanagement practices of crops in terms of date ofsowing, irrigation, and fertilizer application, etc.,due to clearer understanding of various require-ments of plants during different growth stages.This may further help in developing transgenicswith desirable traits. Efforts have been made tounderstand the gene expressions during variousphysiological activities in different plants withthe help of DNA, cDNA and oligonucleotidemicroarrays (Ahroni and Vorst 2001).
For example, (1) expression in roots andleaves (cDNA; 48 clones) (Schena et al. 1995); inmajor plant organs (cDNA; 1,443 clones) (Ruanet al. 1998); response to nitrate treatments(cDNA; 5,524 clones) (Wang et al. 2000); phy-tochrome A mediated response (Oligo; 412polymorphisms) (Spiegelman et al. 2000);expression in developing seeds (cDNA; 2715clones) (Girke et al. 2000); expression in differenttissues, organs, genetic conditions and growthenvironments (Oligo; 8,200 genes represented)(Zhu and Wang 2000) have been studied in Ara-bidopsis. (2) In strawberry, genes expressed forripening and flavor has been identified byemploying cDNA microarray having 1,701clones (Ahroni and Vorst 2001). (3) In Petunia,genes expressed during flower development havebeen studied using cDNA microarray having 480petunia clones (Aharoni et al. 2000). (4) InMaize, expression analysis of the glutathione-
S-transferase gene family has been done withcDNA microarray of 42 maize clones (McGonigleet al. 2000). (5) Similarly, gene expressions indifferent tissues of rice have been studied bycDNA microarray having 1,265 clones.
6.7.3.5 Genetic StudiesAnalysis of gene expressions using DNA micro-arrays may help in ascertaining the phylogeneticrelationship of plant species, races of plant patho-gens and insect pests. Further, it may help indeveloping molecular markers like ESTs and SNPsthat are helpful in tracing the movement of the genein breeding populations while developing newvarieties bearing specific traits. There are fewstudies where cDNA microarrays have been used.
For example, cDNA microarray having 1,152clones has helped in identifying repetitivegenomic fragments in 17 Vicia species thathelped in their phylogenetic resconstruction(Nouzov’a et al. 2001).
6.7.3.6 Multiple Pathogen Detectionand Quantificationof Population Densities
Crop plants are known to be infected with var-ious pathogens in a crop season. It makes it adifficult task to differentiate and identify themultiple pathogens in a single assay at a giventime. DNA microarrays were originally devel-oped for gene expression analysis (Schena et al.1996) but may also be useful in detecting mul-tiple pathogens in a single assay (Lievens andThomma 2005; Lievens and Thomma 2007). Forpathogen detection using DNA microarray, thetarget DNA is amplified using universal primersthose anneal to conserved sequences flanking thediagnostic domains. The amplicons are labeledand then hybridized with the array. Likewise byusing universal primers it is possible to amplifymultiple targets which can be then detected byhybridization to the array. Using this strategyvarious microorganisms like fungi (Lievenset al. 2003; Nicolaisen et al. 2005), oomycetes(Lévesque et al. 1998; Tambong et al. 2006),
6 DNA Microarray 93
bacteria (Fessehaie et al. 2003), nematodes(Uehara et al. 1999) and viruses (Boonham et al.2003) have been successfully detected. Further,to increase the number of multiple detections inone run, combination of sequence-nonspecificamplification techniques with DNA microarraysis found to be useful in detecting the viruses forwhich appropriate universal primers are notavailable. For multiple detections, the discrimi-nation power of DNA microarrays is very highbecause they can discriminate the targets dif-fering even for a SNP if proper hybridizationconditions are maintained (Lievens et al. 2006).
For example, (a) arrays developed to identifyand detect more than 100 species of a fungalgenus Pythium (Tambong et al. 2006). (b) TheDNA array developed for detecting a wide rangeof fungal and bacterial pathogens occurring onhorticultural crops and turf grasses (Lievens andThomma 2005).
Other than multiple pathogen detections,DNA microarray technology is also been founduseful in quantitative determination of microbialpopulation densities because under some situa-tions hybridization signals on the array are foundproportional to the quantity of target DNA in thesample (Lievens et al. 2005). Likewise, it ispossible to detect and quantify multiple patho-gens in a single assay.
For diagnosis of plant pathogens by DNAmicroarray technique, firstly, RNA is extractedfrom infected plant sample which is then con-verted to complementary DNA (cDNA) andsecond-strand synthesis is performed using ran-dom pentamers with an anchor primer sequenceat the 50 end. Likewise due to the use of anchorprimers unbiased amplification of plant and viralRNA takes place, which is then used as a targetfor hybridization of array as shown in Fig. 6.9.
6.8 Designing DNA MicroarrayExperiment
Careful planning of design is crucial to thesuccess of microarray experiments if statisti-cally and biologically valid conclusions are tobe drawn from the data. There are three mainelements to consider when designing a micro-array experiment. First, replication of the bio-logical samples is essential for drawingconclusions from the experiment. Second,technical replicates (two RNA samplesobtained from each experimental unit) help toensure precision and allow for testing differ-ences within treatment groups. The biologicalreplicates include independent RNA extractionsand technical replicates may be two aliquots of
1. PCR amplicon having pathogen and host DNA
3. Application of sample DNA to microarray chip
2. Addition of fluorescent dyes (Red & Green)
4. Hybridization of complementary sequences to microarray
5. Scanning to observe fluorescence
Fig. 6.9 Diagnosis of plant pathogens by DNA microarrays
94 A. M. Charpe
the same extraction. Third, spots of each cDNAclone or oligonucleotide are present as repli-cates (at least duplicates) on the microarrayslide, to provide a measure of technical preci-sion while hybridization. At the same time,importance of replicates cannot be overem-phasized because variability can be very highin microarray experiments. Many groups alsochoose to carry out dye reversals, in which onereplicate array is hybridized with the experi-mental sample labeled with one fluorophoreand the reference sample with the other dye.The corresponding duplicate array is thenhybridized with experimental samples and ref-erence samples labeled with the opposite fluo-rophores. This strategy generates replicate datawhile balancing the possible differential effi-ciency of dye incorporation among RNA sam-ples. It is critical that information about thesample preparation and handling is discussedbefore planing an experiment, in order to helpidentify the independent units in the experimentand to avoid inflated estimates of statisticalsignificance (Churchill 2002).
Microarray experiments are conducted usingthree basic designs (Macgregor and Squire2002):
6.8.1 Case control design
In a case control study, two samples from asingle individual, e.g., tumor and healthy tissuesare compared directly. Because patient vari-ability and genetic heterogeneity are key issuesin microarray data analysis, the case controldesign is an excellent solution to avoid theireffect on results, when feasible.
6.8.2 Blocked Design
Blocked designs are typically used to study theeffect of a treatment or growth condition on asample such as a cell line. They have beensuccessfully used to examine cell lines grownunder different conditions (e.g., cultured in the
presence or absence of an anticancer drug) ordifferent related cell lines (e.g., wild type v/smutant, nontransfected cells v/s transfected cell).
6.8.3 Random Profile Design
Random profile designs are widely used inmicroarray experiments when cell lines orpatient samples are selected and profiled. Mostof the profiling papers have used this design,which offers the ability to use data from manydifferent individuals but offers no intrinsic con-trol for bias in the patient populations or cellpopulations used.
In both the blocked and randomized profiledesigns, the sample is typically compared with acommon or universal reference, which shouldhave adequate representation of the majority ofgenes on the array being profiled and be easilyavailable. Commercially available referenceRNA is often a good choice because of widegene representation (e.g., Stratagene and Clon-tech). The use of a common reference also offersthe advantage of allowing longitudinal compar-ative analysis among several microarray projectsbetween different research groups interested in acommon aspect of cancer research, such astumor progression or resistance to anticancerdrugs (Lashkari et al. 1997)
6.9 Preparation of Targetand Hybridization
Total RNA and mRNA are used for microarrayexperiments that help in generating high-qualitydata with high degree of confidence. For suc-cessful microarray experiments, isolation ofhigh-quality RNA from sample is required.Different standard RNA extraction methodolo-gies have been used successfully. Experimenterscan choose protocol for RNA extraction by theirpersonal experience. Quantitative and qualita-tive evaluation of the RNA obtained can becarried out by standard techniques, such asagarose gel electrophoresis, but it requires
6 DNA Microarray 95
relatively large amounts of RNA samples. Morerecently, assessment of RNA quality and quan-tity has been greatly facilitated by the use ofmicrocapillary-based devices such as the AgilentBioanalyzer (Agilent Technologies), which canbe used with as little as 5 ng of total RNA.
Insufficient availability of RNA is one of themajor limitations in the routine application ofmicroarray technology to disease detection inpatient samples. Thus, there has been consider-able interest in the development of RNA ampli-fication strategies that facilitate RNA extractionfrom laser capture microdissected (LCM) sam-ples, such as fine-needle biopsies. For standardmicroarray experiments, the isolated RNA isreverse-transcribed into target cDNA in thepresence of fluorescent (generally Cy3-dNTP orCy5-dNTP) or radio labeled deoxynucleotides([33P]- or [32P]-a-dCTP) to develop labeled tar-gets. After purification and denaturation, theselabeled targets are hybridized to the microarraysat a temperature determined by the hybridizationbuffer used. After hybridization, the arrays arewashed under stringent conditions to removenonspecific target binding and are air-driedbefore image acquisition and quantification(Macgregor and Squire 2002).
6.10 Imaging and Quantification
Processing of microarray image requires differ-ential excitation and emission wavelengths ofthe two fluors to obtain a scan of the array foreach emission wavelength, typically as two 16-bit grayscale TIFF images. These images arethen analyzed to identify the spots, calculatetheir associated signal intensities, and assesslocal background noise. Most image acquisitionsoftware packages also contain basic filteringtools to flag spots such as extremely low-inten-sity spots, ghosts spots (where background ishigher than spot intensity), or damaged spots(e.g., dust artifacts). These results allow an ini-tial ratio of the evaluated channel/referencechannel intensity to be calculated for every spot
on the chip. The products of the image acquisi-tion are the TIFF image pairing and a quantifieddata file that has not yet been normalized(Macgregor and Squire 2002). Assessment ofdifferent image analysis methods is available onwebsite http://oz.berkeley.edu/tech-reports/.
6.11 Warehousingand Normalization of Data
The quantity of data generated in a microarrayexperiment typically requires a dedicated data-base system to store and organize the microarraydata and images. The first role of a localmicroarray database is the storage and annota-tion (description of experimental parameters) ofmicroarray experiments by the investigator whodesigned and carried out the microarray experi-ments. In addition, there is currently anincreasing global interest in making microarraydata sets publicly available in a standardizedformat. This would allow other investigators toreproduce published microarray experiments,thereby independently verify them, to comparedata sets across different microarray platforms,and importantly, to interrogate publishedmicroarray data sets by use of various bioinfor-matics tools to explore different biologicalproblems. To answer this need, the MinimalInformation About a Microarray Experiment, orMIAME standard, has been proposed by theMGED (http://www.mged.org/) organization asa series of criteria that should be used whendefining microarray experiment parameters. Oneof such database is GeneTraffic of IobionInformatics, which holds all of the microarraydata files and TIFF images, as well as a MIAMEsupportive annotation of experiments (Macgregorand Squire 2002).
Once data have been loaded into the database,they are normalized, and aggregate statistics arecalculated. Normalization is a process that scalesspot intensities such that the normalized ratiosprovide an approximation of the ratio of geneexpression between the two samples. Different
96 A. M. Charpe
strategies are adopted for normalization ofmicroarray data. The choice of a robust andadequate normalization method is as crucial forthe quality of the data obtained as the experi-mental design of the microarray experimentitself. A discussion of normalization methods isavailable on website www.utoronto.ca/cancyto/CLINCHEM.
6.12 Statistical Analysis and DataMining
Microarray data sets are commonly very large,and analytical precision is influenced by a num-ber of variables. Statistical challenges includetaking into account effects of background noiseand appropriate normalization of the data. Nor-malization methods may be suited to specificplatforms and, in the case of commercial plat-forms, the analysis may be proprietary. Algo-rithms that affect statistical analysis include:(a) Image analysis: gridding, spot recognition of
the scanned image (segmentation algo-rithm), removal or marking of poor-qualityand low-intensity features (called flagging).
(b) Data processing: background subtraction(based on global or local background),determination of spot intensities and inten-sity ratios, visualization of data (e.g., MAplot), and log-transformation of ratios, glo-bal or local normalization of intensity ratios.
(c) Identification of statistically significantchanges: t-test, ANOVA, Bayesian method(Ben-Gal et al. 2005), Mann–Whitney testmethods tailored to microarray data sets,which take into account multiple compari-sons or cluster analysis (Priness et al. 2007).These methods assess statistical power basedon the variation present in the data and thenumber of experimental replicates, and canhelp minimize Type I and Type II errors inthe analyses (Wei et al. 2004).
(d) Network-based methods: Statistical methodsthat take the underlying structure of genenetworks into account, representing eitherassociative or causative interactions or
dependencies among gene products (Streiband Dehmer 2008).
Microarray data may require further pro-cessing aimed at reducing the dimensionality ofthe data to aid comprehension and more focusedanalysis (Wouters et al. 2003). Other methodspermit analysis of data consisting of a lownumber of biological or technical replicates;such as the Local Pooled Error (LPE) test poolsstandard deviations of genes with similarexpression levels in an effort to compensate forinsufficient replication (Jain et al. 2003). Geneexpression values from microarray experimentscan be represented as heat maps to visualize theresult of data analysis.
Data mining methods typically fall into oneof two classes: supervised and unsupervised. Inunsupervised analysis, the data are organizedwithout the benefit of external classificationinformation. Hierarchical clustering (Adomaset al. 2008), K-means clustering (Moran et al.2004), or self-organizing maps (Hacia et al.1999) are examples of unsupervised clusteringapproaches that have been widely used inmicroarray analysis (Nuwaysir et al. 2002;Bammler et al. 2005; Adomas et al. 2008).Supervised analysis uses some external infor-mation, such as the disease status of the samplesstudied. Supervised analysis involves choosingfrom the entire data set a training set and atesting set and also involves construction ofclassifiers, which assign predefined classes toexpression profiles. Once the classifier has beentrained on the training set and tested on thetesting set, it can then be applied to data withunknown classification. Supervised methodsinclude k-nearest neighbor classification, sup-port vector machines, and neural nets. Forexample, Shalon et al. (1996) used a k-nearestneighbor strategy to classify the expressionprofiles of leukemia samples into two classes:acute myeloid leukemia and acute lymphocyticleukemia. Tang et al. (2007b) used Large-scaleRNA profiling and supervised machine learningalgorithms to construct a molecular classifica-tion for 10 carcinomas i.e. prostate, lung, ovary,colorectum, kidney, liver, pancreas, bladder/
6 DNA Microarray 97
ureter, and gastroesophagus. Similarly, neuralnetwork analysis has been used by Churchill(2002) to delineate consistent patterns of geneexpression in cancer.
Recently, a strategy called SignificanceAnalysis of Microarrays has been proposed byMicro Array Quality Control (MAQC) project ofNCTR centre for toxicoinformatics, whichallows the determination of significantly differ-entially expressed genes between groups ofsamples analyzed by expression arrays. Thisapproach has been used to narrow down theanalysis to a subset of genes that were also shownto be differentially expressed when analyzed byconventional two-dimensional hierarchical clus-tering. The approach has helped in identifyingthe genes that show differential expressionbetween early-stage epithelial ovarian cancer(EOC), late-stage EOC, and healthy ovary.
6.13 Redundancy and Challenges
DNA microarray technology is a new develop-ing era in gene expression analysis that has itsown limitations and challenges. Few of them areas follows:1. The relation between a probe and the mRNA that
it is expected to detect is not trivial. SomemRNAs may cross-hybridize probes in the arraythat are supposed to detect another mRNA.
2. mRNAs may experience amplification biasthat is sequence or molecule-specific.
3. Probes that are designed to detect the mRNAof a particular gene may be relying ongenomic EST information that is incorrectlyassociated with that gene.
4. Bioinformatics challenges:(a) The multiple levels of replication in exper-
imental design create difficulties in datahandling.
(b) The number of platforms and independentgroups and data format is a challenge forstandardization. Microarray data is difficultto exchange due to the lack of standardiza-tion in platform fabrication, assay protocols,and analysis methods. This presents aninteroperability problem in bioinformatics.
Various grass-roots open-source projectsare trying to ease the exchange and anal-ysis of data produced with nonproprietarychips. For example,
The MIAME checklist helps to define the levelof detail that should exist and is being adoptedby many journals as a requirement for thesubmission of papers incorporating microarrayresults. But MIAME does not describe theformat for the information, so while manyformats can support the MIAME requirements,as of 2007, no format permits verification ofcomplete semantic compliance.The MAQC Project is being conducted bythe US Food and Drug Administration(FDA) to develop standards and qualitycontrol metrics which will eventually allowthe use of micro array data in drug dis-covery, clinical practice and regulatorydecision-making.The MGED Society has developed standardsfor the representation of gene expressionexperiment results and relevant annotations.
(c) The statistical analysis of the data iscomplicated.
(d) Accuracy and precision solely depends onrelation between probe and gene.
(e) To share the sheer volume (in bytes) ofdata dedicated data storage system isrequired.
6.14 Pitfalls of DNA MicroarrayTechnology
Like any other technology, DNA microarraytechnology has some lackings and drawbacks(Vacha 2003). To list a few:
1. Focusing on image quality over data quality.2. Paying more attention to absolute signal
than signal-to-noise.3. Failing to interpret replicate results within
the context of the ‘level’ of replication.4. Assuming that statistical significance is
equivalent to biological significance.5. Ignoring experimental design considerations.6. Using experimental conditions that are dif-
ferent from the error model conditions.
98 A. M. Charpe
7. Paying more attention to the magnitude of thelog ratio than the significance of the log ratio.
8. Automatically assuming that q-pcr, rpa, ornorthern blot analysis is needed to confirmevery microarray result.
9. Cutting upfront costs at the expense ofdownstream results.
10. Pursuing one path in data interpretation.
6.15 Conclusion
DNA microarray is a very useful technology tounderstand the gene functions from sequencesidentified through small or large genomesequencing projects as it helps in handling mul-tiple genes in a go, therefore speeding the func-tional genomics e.g., human genome sequence,rice genome sequence, pigeon pea genomesequence, etc. It is also helpful in understandingthe biochemical details of any physiological stateof an individual, i.e., diseased, stressed, mutated,or constitutive. This technique has tremendouspossibilities in diagnostics encompassing human,animals, plants, birds, etc. Proper experimentalplanning, data warehousing and logical inter-pretation of data may help in developingmolecular medicines for the diseases that are notcurable by conventional medicines. It may alsobe useful to identify valuable genes and genecombinations that may help in developing cropvarieties with improved qualitative and quanti-tative yield traits, to understand genetics, evolu-tion and epidemiology of races of any livingbeing, host responses to biotic and abiotic stres-ses, and drug sensitivity of host and pathogen.Likewise, an in-depth knowledge of many otherbiological phenomenons with practical applica-bility may also be obtained.
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7Metagenomics: The Explorationof Unculturable Microbial World
G. K. Joshi, J. Jugran, and J. P. Bhatt
Abstract
Metagenomics refers to the direct extraction and analysis of the DNAfrom an assemblage of microorganisms for accessing genomic wealth ofmicroorganisms which cannot be cultivated in the laboratory byconventional methods. Given the fact that only 1 % of total microor-ganisms of an environmental sample are actually culturable, advance-ment in metagenomic studies has far increased our access to the genes ofmicrobial community that remained uncultured so far. In today’s worldwhere products of microbial origin have proved their utility in almostevery sphere of life, metagenomic studies have become highly importantas it gives a clue to the hidden wealth of microbial world. This chapterdeals with various aspects of metagenomics.
7.1 Introduction
Studies in microbiology could never be possiblewithout a microscope. So, the event of a humanbeing (Antonie van Leeuwenhoek) seeing firsttime a bacterium in the year 1663 is highlysignificant. Equally appreciable was the work ofbotanist Ferdinand Cohn, who classified many
bacteria and described the life cycle of Bacillussubtilis based on his microscopic observations.Mycologist Franz Unger had understood theconcept of pure culture as early as in 1850s. Butit was in large part the emphasis on diseasecausality that solidified pure culture as thestandard bacteriological technique for laboratorymicrobiology. Robert Koch’s postulates and hisown innovations in developing culture mediawere instrumental in this shift. Microbiologistswere attracted to the power and precision ofstudies of microorganisms in pure culture and asa result, most of the knowledge that fills modernmicrobiology textbooks is derived from theorganisms maintained in pure culture. For a longtime microbiologists focused on discoveriesbased on readily cultured model organismsleading to eruption of knowledge in microbialphysiology and genetics in the last century. Overthe years microorganisms and their products
G. K. Joshi (&) � J. Jugran � J. P. BhattDepartment of Zoology and Biotechnology,HNB Garhwal University, Srinagar (Garhwal),Uttarakhand, Indiae-mail: [email protected]
J. Jugrane-mail: [email protected]
J. P. Bhatte-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_7, � Springer India 2014
105
proved to be useful for mankind in a variety ofways. As a result, a number of industries weresprung up in different parts of the world toharness the benefits of extraordinary versatilemetabolic capacities of these tiny creatures. Inan attempt to isolate and maintain novel micro-organisms, microbiologists across the worldexplored nearly every available ecologicalniche/climatic zone. However, their success wasbased on their ability to develop suitable mediato provide growth support to the desired organ-isms and in this course many of actual inhabit-ants remained uncultured owing to a variety ofcauses like lack of necessary symbionts, nutri-ents, surfaces, excess inhibitory compounds,incorrect combinations of temperature, pressure,or atmospheric gas composition, accumulationof toxic waste products from their own metab-olism, etc. There was paradigm shift in thisapproach as direct analysis of 5S and 16S rRNAgene sequences in the environment were used todescribe the diversity of microorganisms in anenvironmental sample bypassing culturing steps(Pace et al. 1986). The technical difficulties inthis approach were largely overcome with thedevelopment of PCR technology and primerdesigning methods that can be used to amplifyalmost the entire gene. The study of soil bacte-rial diversity, demonstrated with the help ofDNA–DNA re-association techniques revealedthat the complexity of bacterial DNA in the soilwas at least 100-fold greater than that could beaccounted for by traditional culturing approach.This work led to the suggestion that the diversityof uncultured world could far exceed the previ-ous estimates.
It is now estimated that less than 1 % of thetotal microbes in an environment are actuallycultivable through traditional culturing approach(Kimura 2006). Advances in the area of me-tagenomics have enabled us to have access to afar greater number of microorganisms in ahabitat than any time before. Metagenomics is acombination of two separate words—meta andgenomics—derived from the concept of meta-analysis (statistically combining separate analy-ses) and genomics (the wide ranging analysis ofan organism’s genetic material) as defined by
Schloss and Handelsman in 2003. It can bereferred to as the culture-independent analysis ofmicrobial communities. It involves the directisolation of genomic DNA from an environmentfollowed by its cloning in easily cultivablemicroorganisms (Fig.7.1). The approach thusobviates the need for culturing the microorgan-ism in order to have access to its genomicwealth. There are other terms used for the sameapproach as community genomics, environ-mental genomics, and population genomics.Pace et al. (1985) first proposed the thought ofcloning DNA directly from environmentalsamples and the report for such cloning in phagevector appeared 6 years later (Schmidt et al.1991). Over the years, following the initialreport, culture-independent analysis have madeit apparent that a large number of ‘yet to becultivated’ bacteria belong to phylogenticallydistinct genotypic classes.
7.2 Steps in Metagenomic Analysis
7.2.1 DNA Isolation
In general, metagenomic DNA isolation strategyshould result in representation from a broadrange of organisms. DNA isolation is, therefore,the most important step for the subsequentdownstream genome analysis. Shearing of DNAshould be avoided during extraction as longintact fragments are required for communityanalysis. Further, the DNA should be free fromcontaminating elements for easy downstreamstudies. It is difficult to develop general protocolfor DNA isolation as the environmental samplemay contain enormous group of microorganismsbelonging to different genotypes, classes, anddivisions having marked difference in their cellwall structure. In addition to that, problem forrecovery of DNA straight from environmentalsample lies in the fact that cells are in a physi-ological state that is hard to lyse. Designing of aunique ideal DNA isolation protocol for recov-ery from every microbial cell in the sample is,therefore, extremely difficult. Despite everyeffort certain groups are represented more in
106 G. K. Joshi et al.
terms of the isolated genomic material comparedto others. However, in certain cases a genomicbiasness is deliberately introduced where theaim is to collect DNA from certain group ofbacteria with some desirable properties. Thereare two main strategies for extraction of me-tagenomic DNA viz. ‘direct method’ involvingin situ cell lysis and ‘indirect method’ wherecells are first separated from the sample and thenlysed. Both methods are equally good as far asthe DNA analysis for determination of diversityspectrum is concerned. However, the formermethod results in more amount of DNA in animpure form. In case of soil the problem ofcontaminating agents such as humic acid mayinterfere with the process of DNA isolation. Onthe other hand, a large amount of water has to beprocessed in order to have a significant yield ofDNA from aquatic samples.
Vigorous cell lysis methods are generallyemployed considering the equal representationof each community member from the diversepopulation in an environmental sample. Thismay, however, lead to the shearing of genomicfragments and thus making the intact generecovery difficult. Also, there is an increased riskof chimera formation from small template DNAduring subsequent PCR. Extraction of total me-tagenomic DNA is, therefore, a compromisebetween the vigorous extraction and quality ofDNA required for the representation of allmicrobial genomes. Other physical methods forDNA isolation include freeze-thawing, ultra-sonication, etc. Chemical methods, on the otherhand, are preferred when the aim is to selectcertain taxa by exploiting their unique bio-chemical properties. SDS-based cell lysis is themost common type of method for release ofintracellular DNA. Mechanical method is shownto recover more diversity than the chemicalmethod. A combination of different physical andchemical methods can also be used dependingon the soil characteristics and associatedmicrobial diversity.
Since not all members of a community areevenly represented in the extracted genome,experimental normalization is sometimes nec-essary as a partial solution to it. One such
approach involves density gradient separation ofgenotypes in the presence of an intercalatingagent. This leads to the separation of genomesbased on their % G and C content as distinctbands in centrifugation tube. A normalized me-tagenome is then obtained by combining equalamount of each band. Another method for nor-malization for rare sequences involve denaturingmetagenomic DNA followed by re-annealingunder stringent conditions. Compared to raresequences, abundant single-stranded DNA willanneal rapidly and acquire double-strandedform. Single-stranded sequences can then beseparated from the double-stranded nucleicacids.
A precultivation step improves the quality ofthe community DNA. This, of course, reduces thechance of representation of maximum diversity ofthe original environmental sample and enhancesthe possibilities of recovering genomes frommicroorganisms with desired trait. The importantstrategies that have been employed for enrichingthe metagenome for sequences of interest beforecloning, are summarized in Table 7.1. Other latestmethods for enrichment are based on microarray,suppressive subtractive hybridization, differentialexpression analysis, and multiple displacementamplification.
7.2.2 Library Construction and Choiceof Vector
Metagenomic library has been created in almostall types of commonly used gene cloning vectorssuch as plasmid, phage, cosmid, fosmid, bacte-rial artificial chromosome (BAC), etc. Duringthe early studies plasmid-based vectors wereprimarily used for metagenomic library with lessthan 10 kb insert size. Large capacity vectors arenow fast replacing the plasmid-based vectors.The choice, however, depends on the overall aimof the study. Small insert library is suitable forcloning of individual gene. These libraries areuseful for functional screening of clonesexpressing various enzymes like chitinase,lipase, esterase, amylase, nitrilase, etc. It alsoprovides large amount of sequencing
7 Metagenomics: The Exploration of Unculturable Microbial World 107
information. The main drawback with smallcapacity vectors is the huge number of clones inthe library which are sometimes very difficult tohandle. On the other hand, large insert library isrequired when gene cluster or operons are to beisolated and studied. Cosmid or fosmid vectorshave been used to create library with an insertsize of about 40 kb, whereas BAC vectors canmarkedly reduce the size of the metgenomiclibrary by cloning up to 150 kb. The recovery ofhigh molecular weight DNA is, however, aprerequisite for using the high capacity vectors,which is sometimes very difficult to achieve asthe soil compositions demand vigorous isolationstrategies leading to extensive fragmentation ofDNA. Escherichia coli is the most preferredchoice of host. Other hosts such as Streptomyceslividans, Pseudomonas putida, and Rhizobiumsp. have also been successfully used.
7.2.3 Screening Strategies
7.2.3.1 Function BasedIn a metagenomic library clones with usefultraits can be traced by mainly two methods viz.function-based screening and sequence-basedscreening. The functional screening depends onthe faithful expression of the cloned gene in aheterologous host. Since no pre-sequence
analysis of the gene of interest is required, thereis very high chance to identify entirely newclasses of genes for new or known functions.The expression of the gene may confer someselective advantage to the desired clone makingits visual identification possible in a pool ofclones. For example, antibiotic resistance genesor enzyme producing genes can be selected onspecific plates (Fig. 7.2). However, very fewclones in a metagenomic library express anygiven activity. Many thousand clones need to bescreened before getting one with desirable trait.In the recent years highly efficient screening andselection systems have been developed forselecting positive clones. Uchiyama et al. (2005)have developed Substrate-induced gene expres-sion screening (SIGEX) for rapid identificationof clones showing catabolic gene expression.The technique is based on the fact that catabo-lite-gene expression requires certain substratesas inducers which, in many cases, is controlledby regulatable elements situated near to cata-bolic genes. A plasmid-based operon-trapexpression vector (p18GFP) containing gene forgreen fluorescent protein (GFP) immediatelyafter the insert site, is constructed and used forcreating metagenomic library in this technique.The clones are exposed to an inducer whicheasily diffuses to the cyptoplasm of all trans-formed cells in the library. In positive clones(in which the entire operon responding to that
Table 7.1 Metagenomic library enrichment methods
Enrichmentmethod
Principle References
Based on GCcontent
Enrichment for the sequences of interest by its separation from the bulkmetagenomic DNA based on the variation in GC contents throughultracentrifugation.
Schloss andHandelsman (2005)
BrdU enrichment Incorporation of bromodeoxyuridine (BrdU) in the soil leading to itsincorporation into the DNA of metabolically active subset of thecommunity. The labeled DNA can then be separated byimmunocapture technique
Urbach et al.(1999)
Stable isotopeprobing (SIP)
C13 labeled substrate is provided to the soil bacteria and those capableof metabolizing the substrate incorporate the isotope into their DNA.The heavier DNA due to C13 is separated from the bulk by densitygradient centrifugation
Radajewski andMurrell (2002)
Cultureenrichment
The sample can be enriched for desired microbes by varyingnutritional, physical, and chemical conditions during precultivationstep
Singh et al. (2009)
108 G. K. Joshi et al.
substrate/inducer is cloned), the inducer binds tothe cloned regulatory elements and causesexpression of downstream sequences for GFP.The GFP positive cells can then be separated byfluorescence activated cell sorter (FACS).Another similar approach is METREX in whichproduct of metagenomic DNA induces bacterialquorum sensing. The metagenomic library iscreated in host cells which already contain areporter plasmid. The reporter plasmid containsa reporter gene such as gfp under the control of aregulatory promoter. The promoter can beupregulated in the presence of the product whosegene is present only in the positive clones in thelibrary. The positive clones thus express gfp andcan be identified and separated from other clonesin the library.
7.2.3.2 Sequence BasedThis strategy is based on the prior information oftarget gene sequence. The classical approach ofhybridization with labeled probe can be used toidentify the positive clones in the metagenomiclibrary (Fig. 7.3). Complete sequencing of clonesthat contains phylogenetic anchors indicates thetaxonomic group of the source of DNA. Randomsequencing can also be done to identify a gene ofinterest followed by assessment of flanking DNAto contain any phylogenetic anchor. The power-ful approach of sequence analysis guided by theidentification of phylogenetic markers producedthe first genomic sequence linked to a 16S rRNAgene that affiliated with c–Proteobacteria(Handelsman 2004). Sequencing random clonesis an alternative to phylogenetic marker-based
DNA extraction
DNA fragmentation and size
separation
Genomic library
preparation
Analysis of clones in the
genomic library
Collection of
environmental
samples
Fig. 7.1 Steps inmetagenomic analysis
7 Metagenomics: The Exploration of Unculturable Microbial World 109
approach. The field of metagenomics has beentransformed by the application of a whole gen-ome shotgun sequencing approach during the last6–7 years. Next-generation ultra high-through-put sequencing techniques have revolutionizedthe field with heavy price cut allowingsequencing at scale larger then anytime before.Sequencing projects have been carried out toassess the actual microbial diversity and theirecological inference in different environmentsuch as sea and acid mine drainage, etc. In thepast few years, techniques of DNA microarrayhas also been widely used for screening clones inmetagenomic library.
The technique of PCR has been applied to theisolation and detection of novel genes fromcommunity genome. However, the major limi-tation with PCR is to rely on the flanking DNAsequence information for isolation of anunknown gene from an uncultured organism.
Furthermore, when PCR is directed at conservedsequence motifs, only partial genes are recov-ered. In an attempt to solve this problem, a novelstrategy for recovering complete open readingframe from environmental DNA samples wasproposed by Stokes in 2001. His team designedPCR assays targeted toward the 59-base elementfamily of recombination sites that flank integ-rons associated gene cassettes. This approachhas resulted in the amplification of diverse genecassettes containing complete open readingframe from the environmental DNA.
7.3 Applications of Metagenomics
Metagenomic studies are important and have arange of applications for both academia andindustries. The recent findings in metagenomicssuggest the role of microorganism in influencing
Positive clone
Fig. 7.2 Function-basedvisual identification ofpositive clone on solidmedium
Labeled probe
Positive clone
generates signal
No signal with
undesirable clones
Fig. 7.3 Sequence-basedidentification of positiveclone
110 G. K. Joshi et al.
human life far beyond the previous expectations.The important applications and outcome fromthese studies are summarized below.
7.3.1 Ecological Inferencesfrom Microbial DiversityEstimation
In microbial ecology it is important to know howmicroorganisms acquire nutrients and produceenergy, form symbioses and compete, andcommunicate with other members of the com-munity. Metagenomic investigations haveresulted in the identification of various novel lifeforms in geographically distinct region andattempts are still underway to describe theirpossible role in that environment. As definedbelow metagenomic findings have resulted inrestructuring of our understanding of the eco-logical balance in most of the environments.
7.3.1.1 Community StructureGenomic information of the ‘unculturable’ bac-teria can help in understanding their physiologyand also their role in ecosystems. Metagenomicinvestigation has revealed that bacteriorhodop-sins that function as light- driven proton pumpfor energy generation in halophilic archaea arealso present in eubacteria. Presence of rhodopsinin marine proteobacteria suggested the possi-bility of a new phototrophic pathway that mayinfluence the flux of carbon and energy in theocean’s photozone worldwide. By utilizingshotgun sequencing approach complete genomesof Leptospirillum group II and Ferroplasma typeII were assembled from a natural acidophilicbiofilm. Pathways of carbon and nitrogen fixa-tion and energy generation for every organism ofthe simple biofilm community were established.The almost complete assembly of the genome ofan uncultured bacterium, Kuenenia stuttgarti-ensis, has revealed unique metabolic adaptationsassociated with anaerobic ammonium oxidation.PCR-DGGE (Denaturing gradient gel electro-phoresis) is a sophisticated method that enablesus to visualize a complete microbial community
as a simple banding pattern and also allows toeasily monitor community dynamics within theenvironment. In addition to usual 16S rRNA,various functional genes have also been targetedto investigate the diversity and communitystructure in this approach. Sargasso Sea genomeproject carried out by Venter et al. (2004) hasresulted in the identification of genes for trans-port of phosphonates and utilization of poly-phosphates and pyrophosphates. The findingindicates the microbial community’s capabilityto survive in the extremely phosphate-limitedenvironment of Sargasoo Sea.
7.3.1.2 Diversity AnalysisThe present barriers in estimation of microbialbiodiversity are largely overcome by the devel-opments in the area of metagenomics. A largeproportion of uncultivated bacteria belonging tonew genotypes, classes, and divisions in thedomains eubacteria and archaea have been dis-covered so far through this approach. Bacteriathose were previously unknown in most well-characterized samples like dental plaques, seawater, and garden soil have been identified.Unlike previous assumptions presence of ar-chaea in habitats like soil, seawater, etc., hasbeen noticed. Sargasso Sea genome project hasled to identification of 1.2 million previouslyunknown genes and 148 previously unknownphylotypes (Venter et al. 2004). Metagenomicstudies have also revealed important informationabout the viral diversity of the human andmarine environments. Breitbart et al. (2002)after studying genomic analysis of two uncul-tured marine viral communities reported thatover 65 % of the sequences were not signifi-cantly similar to previously reported sequences.The most common significant hits among theknown sequences were found belonging toviruses. In the recent years, phage diversity hasalso been revealed by metagenomic technology.
7.3.1.3 SymbiosisMany of the bacteria which have intimateassociation with their eukaryotic hosts
7 Metagenomics: The Exploration of Unculturable Microbial World 111
particularly in the marine environment cannot becultivated in the laboratory. Metagenomics pro-vides a way to reconstruct their genome, thusleading insight into the evolution of the symbi-osis between the host and the bacterium. The firstgenome reconstruction of an uncultured organ-ism was that of Buchnera aphidicola, the endo-symbiont of aphids (Handelsman 2004). Sincethen various other bacterial partners of differenteukaryotic hosts have been studied through me-tagenomic approach. The study becomes impor-tant in view of the fact that a number ofbiomedically relevant natural products like anti-cancer agents, anti-infective agents of otherbioactivities are obtained from invertebrate ani-mals, e.g., sponges, tunicates, and bryozoans andthere is increasing evidence that many of thesemetabolites are produced by these animals inassociation with their bacterial symbionts.
7.3.1.4 PaleogenomicsAnother application of metagenomics has beensuggested in the area of paleogenomics where ithas been useful for resolving phylogeneticrelationships between extinct and modern ani-mals. PCR-based amplification of mitochondrialDNA strategies were also used to set the missinglink. It is assumed that over the years the ancientanimal DNA got mixed with the genomes of theabundant opportunistic microbes. A metage-nomic approach has been defined to obtain cavebear sequence and homology search with PCRamplified genes from black, brown, and polarbears verified the origin and a phylogenetic treewas constructed (Singh et al. 2009). The resultsof such novel strategies suggest the active needfor initiatives to take up genome projects tar-geting extinct species to revolutionize the areaof paleobiology.
7.3.2 Biotechnological Prospectsof Metagenomics
The usefulness of the microbes has been har-nessed by mankind in the form of billion dollarindustries utilizing these tiny organisms for the
production of a variety of products. The advan-ces in metagenomics have revealed that what weknow today is just a tip of the iceberg. Manymore novel gene(s) and their products are yet tobe discovered in near future. Metagenomicsprovides a way to peep into this hidden wealth ofmicrobial world. Following are some of theareas important from biotechnological point ofview, where metagenomics can revolutionize theexisting trends for industrial production ofmetabolites.
7.3.2.1 Bioactive CompoundsNatural products have been a productive sourceof lead structures for designing and developingnew antimicrobial substances. Metagenomicsprovides a way for cloning the biosynthetic geneclusters of bioactive natural products, whichmay in turn provide a renewable source ofcompounds that have often been difficult toisolate in sufficient quantities to permit extensivebiological testing. Pederin, an anticancer agentoriginally isolated from the beetle Paederusfuscipes is synthesized by cooperative activity ofmany genes. The biosynthetic gene cluster for itwas recovered from a beetle derived metage-nomic DNA library that was later found tooriginate from an uncultured symbiotic Pseu-domonad. Clones that produce proteins withpotential anti-infective properties have also beenidentified through metagenomics. A range ofnovel antibiotics have been detected in metage-nomic libraries. Brady et al. (2001) have repor-ted the production of deoxyviolacein and a broadspectrum antibiotic violacein from a soil me-tagenomic library. In an investigation two-col-ored triaryl cation antibiotics, i.e., TurbomycinA and Turbomycin B and their genes have beenisolated from a soil metageomic library(Gillespie et al. 2002). Genes for vitamins likebiotin and vitamin C have also been detected inmetagenomic library.
7.3.2.2 Antibiotic Resistance GeneMetagenomic libraries have also been exploredfor isolation of antibiotic resistance genes from
112 G. K. Joshi et al.
environmental samples. This may give a majorinsight into the mechanism of microbial drugresistance. In today’s world where developmentof antibiotic resistance by pathogens has becomea major concern for health practitioners andpharmaceutical industries, metagenomic studieshave given a vital clue in terms of the discoveryand identification of novel antibiotic resistancegenes. Identification of 9 clones expressingresistance to aminoglycoside antibiotics and oneexpressing resistance to tetracycline from a soilmetagenomic library has been reported byRiesenfeld et al. (2004). Environment like humanoral cavity has been found to be a source oftetracycline, amoxicillin, and gentamycin resis-tance genes. The search for novel anti-microbialgenes and compounds is probably the field that isadvancing most rapidly in metagenomics.
7.3.2.3 Novel EnzymesA wide range and a remarkable number of bio-catalysts have been discovered from uncultur-able microbes. This may be attributed to the factthat gene expressed in nature represents proteins(here enzymes) which presumably, through theevolutionary process has been undergoing hardand long selection pressure. The pressure thusexerted drives the gene evolution towardenzyme production which is most fit for solvinginteraction with the substrate needed for a givenorganism in an ecological niche it is adapted toinhabit. The single most biotechnologicalapplication of metagenomic investigations hav-ing the largest number of publications world-wide so far can be seen in the area ofbiocatalysts. There are more than 100 publica-tions for a single class of enzyme like lipaseproduction through metagenomic clones. In asimilar manner, a vast number of reportsworldwide indicate the possibility of identifica-tion and isolation of novel enzymes and theirgenes through metagenomic approach. There areseveral reports of isolation of novel variants formajority of known industrial enzymes likelipase, protease, amylase, chitinase, cellulase,
xylanase, agarase, nitrilase, etc., from metage-nomic library created from different types of soilor aquatic samples. Table 7.2 enlists some of thelatest publications in just last 3 years reportingnovel enzyme identification through metage-nomic library.
7.4 Role of Bioinformaticsin Developmentof Metagenomics
Developments in the area of bioinformatics havea marked role in establishing the approaches ofmetagenomics to the current level. Manypromising advances in terms of correct analysisand interpretation of vast data generated bymetagenomic studies across the globe havecome from the bioinformatics people. Specificcomputational and statistical tools have beendesigned for metagenomic data analysis andcomparison. Programs like MetaGene and Orp-helia are available for gene prediction for shortreads of around 700 bp. MEGAN, MLTreeMap,AMPHORA, and CARMA are similarity-basedand phylogeny-based phylotyping tools thatutilize similarity searches of the metagenomicsequence against a database of knowngenes/proteins. ARB is an interactive softwaretool for sequence database (RNA/DNA/aminoacids). MG-RAST is an automated server thatfacilitates automatic phylogenetic and functionalanalysis of genome. PHACCS is an online toolfor estimating the structure and diversity ofuncultured viral communities using metage-nomic information. AMOVA and HOMOVA arestatistical tools for genetic diversity estimationamong members of different communities in ametagenome. A tool like DOTUR has beendeveloped for analysis of community member-ship and structure comparison. It is used todetermine whether a metagenomic library con-tains enough number of genes for it to be con-sidered representatives of the diversity in theoriginal microbial community.
7 Metagenomics: The Exploration of Unculturable Microbial World 113
7.5 Conclusion and FutureProspects
Metagenomics is a study of uncultivated micro-organisms in the environment. It is expected thatthe number of novel genes identified using thisapproach will soon exceed the number identifiedthrough sequencing of novel genes from isolatedindividual microbes. It has potential to provideinsight into the functional dimensions of envi-ronmental genomic datasets. Although the out-comes of metagenomic studies worldwide haverefined some of our current understanding ofmicrobial diversity and its possible interactionwith the nature, it is too early to describe it as‘come of age’ technology. Finding a useful genethrough functional screening is still like searchinga needle in a haystack as thousands of clones areto be screened in order to find a positive one.Every year new approaches and technical inno-vations in this area are reported but many of thetechnical difficulties are yet to be fully resolved.Better designing of vectors and perfect host arerequired to overcome this problem. Similarly,steps of gene enrichment, DNA extraction, libraryconstruction, and phylogenetic affiliation of iso-lated genes need to be improved to yield optimumresults. The upgradation and sophistication ofsequencing techniques are bound to revolutionizethis area by enabling scientists to take up Gb-scale metagenomic projects for sequencing
complex communities to (nearly) saturation.However, much deeper sampling methods alongwith special assembly, analysis tools, and datastorage infrastructure are required before suchprojects are initiated. Metatranscriptomics andmetaproteomics are the complementary approa-ches that are seen with great expectation to pro-vide promising results in near future.
References
Bayer S, Kunert A, Ballschmiter M, Greiner-Stoeffele T(2010) Indication for a new lipolytic enzyme family:isolation and characterization of Two esterases from ametagenomic library. J Mol Micobiol Biotechnol18(3):181–187
Bayer S, Birkemeyer C, Ballschmiter M (2011) Anitrilase from a metagenomic library acts regioselec-tively on aliphatic dinitriles. Appl Microbiol Bio-technol 89(1):91–98
Bhuiyan FA, Nagata S, Ohnishi K (2011) Novel chitinasegenes from metagenomic DNA prepared from marinesediments in southwest Japan. Pak J Biol Sci14(3):204–211
Brady SF, Chao CJ, Handelsman J, Clardy J (2001)Cloning and heterologous expression of a naturalproduct biosynthetic gene cluster from cDNA. OrgLett 3:1981–1984
Breitbart M, Salmon P, Andresen B, Mahaffy JM, SegallAM, Mead D, Azam F, Rohwer F (2002) Genomicanalysis of uncultured marine viral communities. ProcNatl Acad Sci USA 99:14250–14255
Couto GH, Glogauer A, Faoro H, Chubatsu LS, SouzaEM, Pedros FO (2010) Isolation of a novel lipasefrom a metagenomic library derived from mangrovesediment from the south Brazilian coast. Genet MolRes 9(1):514–523
Table 7.2 Metagenomic discovery of novel enzymatic genes in recent years
S.No. Enzyme name Source References
1. Esterase Sheep rumen Bayer et al. (2010)
2. Alginate lyase Gut microflora of abalone Sim et al. (2012)
3. Cellulase Soil Liu et al. (2011)
4. Amylase Soil Sharma et al. (2010)
5. a-amylase Soil Vidya et al. (2011)
6. Protease Desert land Neveu et al. (2011)
7. Monooxygenase Industrial effluent sludge Singh et al. (2010)
8. Laccase Soil Ye et al. (2011)
9. Chitinase Marine sediment Bhuiyan et al. (2011)
10. Nitrilase Oil contaminated soil Bayer et al. (2011)
11. Dichlorohydroxylase Pesticide contaminated soil Lu et al. (2011)
12. Lipase Mangrove sediment Couto et al. (2010)
114 G. K. Joshi et al.
Gillespie DE, Brady SF, Bettermann AD, Cianciotto NP,Liles MR, Rondon MR, Clardy J, Goodman RM,Handelsman J (2002) Isolation of antibiotics Turbo-mycin A and B from a metagenomic library of soilmicrobial DNA. Appl Envion Micorobol 68(9):4301–4306
Handelsman J (2004) Metagenomics: application ofgenomics to uncultured microorganisms. MicrobiolMol Bio Rev 68(4):669–685
Kimura N (2006) Metagenomics: access to unculturablemicrobes in the environment. Microbes Environ21(4):201–215
Liu W, Shen W, Cao H, Cui Z (2011) Cloning andfunctional characterization of a novel endo-b-1,4-glucanase gene from a soil-derived metagenomiclibrary. Appl Mirobiol Biotechnol 89(4):1083–1092
Lu Y, Yu Y, Zhou R, Sun W, Dai C, Wan P, Zhang L,Hao D, Ren H (2011) Cloning and characterization ofa novel 2,4-dichlorophenol hydroxylase from ametagenomic library derived from polychlorinatedbiphenyl-contaminated soil. Biotechnol Lett33:1159–1167
Neveu J, Regard C, Dubow MS (2011) Isolation andcharacterization of two serine proteases from me-tagenomic libraries of the Gobi and Death Valleydeserts. Appl Microbiol Biotechnol 91(3):635–644
Pace NR, Stahl DA, Lane DJ, Olsen GJ (1985) Analyzingnatural microbial populations by rRNA sequences.ASM News 51:4–12
Pace NR, Stahl DA, Lane DJ, Olsen GJ (1986) Theanalysis of natural microbial populations by ribo-somal RNA sequences. Adv Microb Ecol 9:1–55
Radajewski S, Murrell JC (2002) Stable isotope probingfor detection of methanotrophs after enrichment with13CH4. Methods Mol Biol 179:149–157
Riesenfeld CS, Goodman RM, Handlesman J (2004)Uncultured soil bacteria are a new reservoir of newantibiotic resistance gene. Environ Microbiol6:981–989
Schloss PD, Handelsman J (2003) Biotechnologicalprospects from metagenomics. Curr Opn Biotechnol14:303–310
Schloss PD, Handelsman J (2005) Introducing DOTUR, acomputer program for defining operational taxonomicunits and estimating species richness. Appl EnvironMicrobiol 71(3):1501–1506
Schmidt T, DeLong EF, Pace NR (1991) Analysis of amarine picoplankton community by 16S rRNA genecloning and sequencing. J Bacteriol 173:4371–4378
Sharma S, Khan FG, Qazi GN (2010) Molecularcloning and characterization of amylase from soilmetagenomic library derived from NorthwesternHimalayas. Appl Microbiol Biotechnol86:1821–1886
Sim SJ, Baik KS, Park SC, Choe HN, Seong CN, ShinTS, Woo HC, Cho JY, Kim D (2012) Characteriza-tion of alginate lyase gene using a metagenomiclibrary constructed from the gut microflora ofabalone. J Industrial Microbial Biotechnol39(4):585–593. doi:10.1007/s10295-011-1054-0
Singh J, Behal A, Singla N, Joshi A, Birbian N, Singh S,Bali V, Batra N (2009) Metagenomics: concept,methodology, ecological inference and recentadvances. Biotechnol J 4:480–494
Singh A, Chauhan N, Thulasiram HV, Taneja V, SharmaR (2010) Identification of two flavin monooxygenasesfrom an effluent treatment plant sludge metagenomiclibrary. Biores Technol 101(21):8481–8484
Stokes HW, Holmes AJ, Nield BS, Holley MP, Nevalai-nen KMH, Mabbutt BC, Gillings MR (2001) Gene:cassette PCR: sequence independent recovery ofentire genes from environmental DNA. Appl EnvironMicrobiol 67:5240–5246
Uchiyama T, Abe T, Ikemura T, Watanabe K (2005)Substrate induced gene expression screening ofenvironmental metagenomic libraries for isolation ofcatabolic genes. Nature Biotechnol 23(1):88–93
Urbach E, Vergin KL, Giovannoni SJ (1999) Immuno-chemical detection and isolation of DNA frommetabolically active bacteria. Appl Environ Micro-biol 65:1207–1213
Venter JC, Remington K, Heidelberg JF, Halpern AL,Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE,Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW,Nealson K, White O, Peterson J, Hoffman J, ParsonsR, Tillson HB, Pfannkoch C, Rogers YH, Smith HO(2004) Environmental genome shotgun sequencing ofthe Sargasso sea. Science 304:66–74
Vidya J, Swaroop S, Singh SK, Alex D, Sukumaran RK,Pandey A (2011) Isolation and characterization of anovel a-amylase from a metagenomic library ofWestern Ghats of Kerala, India. Biologia 66(6):939–944
Ye M, Li G, Qu W, Liu YH (2011) Molecular cloningand characterization of a novel metagenome-derivedmulticopper oxidase with alkaline laccase activity andhighly soluble expression. Appl Microbiol Biotechnol87(3):1023–1031
7 Metagenomics: The Exploration of Unculturable Microbial World 115
8Proteomics
Indu Ravi
Abstract
Proteins are macromolecules and the main components of the physio-logical metabolic pathways of cells. The post-genomic era focuses on thecomplete understanding of the structure and functions of proteins.Proteome, the protein complement of a given cell at a specific time is themain focus of proteomics. Proteomics has recently been of interest toscientists because it gives a much better understanding of an organismthan genomics. A number of advanced techniques such as 2D-gelelectrophoresis, HPLC, Mass spectrometry, MALDI-TOF, NMR, DNAmicroarray, etc., are used for the study of proteomics. The mostimportant applications of proteomics are in the discovery of biomarkersand drug targets. It will also be useful in diseases which involvemultigenes, e.g., cancer, diabetes II, asthma, hypertension, cardiovascularand neurological diseases. This chapter focuses on the basic stepsinvolved, techniques used, and applications of proteomics.
8.1 Introduction
Within a given human proteome, the number ofproteins can be as large as 2 million. Proteinsthemselves are macromolecules, i.e., long chainsof amino acids which are constructed when thecellular machinery of the ribosome translatesRNA transcripts from DNA in the cell’s nucleus.The transfer of information within cells is com-monly from DNA to RNA to protein (Fig. 8.1).
Proteins perform vital functions in the bodysuch as:• Catalyzing various biochemical reactions
through enzymes.• Acting as messengers through neuro
transmitters.• Acting as control elements which regulate cell
reproduction.• Influencing growth and development of vari-
ous tissues through trophic factors.• Transporting oxygen in the blood through
hemoglobin.• Defending the body against diseases through
antibodies.The proteome changes constantly in response
to tens of thousands of intra- and extracellularenvironmental signals. The proteome varies with
I. Ravi (&)Indira Gandhi National Open University (IGNOU),Regional Centre Jaipur, 70/79, Patel Marg,Mansarovar, Jaipur, Rajasthan 302020, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_8, � Springer India 2014
117
health or disease, the nature of each tissue, thestage of cell development and effects of drugtreatments. As such, the proteome often isdefined as ‘‘the proteins present in one sample(tissue, organism, cell culture) at a certain pointin time.’’
The sequencing of the human genome hasprovided comprehensive resources for genomicdata. In addition, the numerous fields such astranscriptomics, proteomics, and metabolomics,etc., and systems biology are providing newways to study living processes. Genomics pro-vides an overview of the complete set of geneticinstructions provided by the DNA, while trans-criptomics looks into gene expression patterns.Proteomics studies dynamic protein productsand their interactions, while metabolomics isalso an intermediate step in understandingorganism’s entire metabolism (Fig. 8.2).
Proteomics attempts to study the structure,function, and control of biological systems andprocesses by systematic and quantitative analy-sis of proteins. The term ‘‘proteomics’’ was firstcoined in 1997 to make an analogywith genomics, the study of the genes. The word‘‘proteome’’ is a blend of ‘‘protein’’ and ‘‘gen-ome’’, and was coined by Marc Wilkins in 1994while working on the concept as a Ph.D. student.Different definitions of proteomics have been
given by different workers from time to time as,the study of full set of proteins encoded by thegenome or the study of all the proteins expressedin a cell.
As the genome describes the genetic contentof an organism, a proteome defines the proteincomplement of the genome. The proteome isdynamic, and is the set of proteins expressedin a specific cell, given a particular set of con-ditions. Proteomics begins with the functionallymodified protein and works back to the generesponsible for its production.
8.1.1 Structure of Proteins
Proteins can be organized in four structurallevels:1. Primary—The amino acid sequence, con-
taining members of 20 amino acids.2. Secondary—Local folding of the amino acid
sequence into a helices and b sheets.3. Tertiary—3D conformation of the entire
amino acid sequence.4. Quaternary—Interaction between multiple
small peptides or protein subunits to create alarge unit.Each level of protein structure is essential to
the biological function of the protein. The primary
Replication DNA
Transcription
mRNA
TranslationProteins
Fig. 8.1 Information transfer in the central dogma of biology
Phenotype
Fig. 8.2 Relationshipbetween genomics,transcriptomics,proteomics, andmetabolomics
118 I. Ravi
sequence of the amino acid chain determineswhere secondary structures will form, as well asthe overall shape of the final 3D conformation.The 3D conformation of each small peptide orsubunit determines the final structure and functionof the protein (Garret and Grisham 1995).
The proteome is the entire complement ofproteins. It is now known that mRNA is notalways translated into protein, and the amount ofprotein produced for a given amount of mRNAdepends on the gene it is transcribed from andon the current physiological state of the cell.Proteomics confirms the presence of the proteinand provides a direct measure of the quantitypresent. The level of transcription of a genegives only a rough estimate of its level ofexpression into a protein. A mRNA produced inabundance may be degraded rapidly or trans-lated inefficiently, resulting in a small amount ofprotein. Many proteins experience post-
translational modifications that profoundly affecttheir activities and many transcripts give rise tomore than one protein, through alternativesplicing or alternative post-translational modifi-cations. Further, many proteins form complexeswith other proteins or RNA molecules, and onlyfunction in the presence of these other moleculesand finally, protein degradation rate plays animportant role in protein content.
8.1.2 Broad-Based Proteomics
The first step when utilizing broad-based pro-teomics is to develop a hypothesis specific to theproteome being studied. For this an organism thatalready has a great deal of genomic informa-tion available is taken, since the genome isalways a useful supplement to proteomic infor-mation. Then the technologies are chosen which
Table 8.1 Broad-based proteomics approach versus traditional focused approach
Broad-based approach Focussed approach
Goal Understand the proteome as a whole Understand specific protein function
Basic steps 1. Identify organism 1. Identify protein
2. Understand sample type and preparation 2. Understand sample type and preparation
3. Utilize analytical technology compatiblewith sample type
3. Isolate protein
4. Bioinformatic analysis of the proteomesample
4. Utilize analytical technology that iscompatible
5. Build a proteomic model 1. Bioinformatic analysis of the proteinsample
2. Model protein’s function and/or structure
Pros 1. Proteomic information about a specifictissue under certain conditions can be gained
1. Inexpensive and results can be generatedmuch quicker than a large proteomic study
2. Relationships between many proteins canbe understood
1. Functional and structural informationabout a protein can be determined
Cons 1. Extensive upfront planning 1. Protein information may not be valuablewithout a global proteomic understanding
2. The study will cost more and last longerthan a focused study
2. Hard to develop global relationships frommany focused experiments taken together
3. No guarantee that quality proteomic datawill be generated in the end
Commontechnologiesimplemented
1. SDS-PAGE 1. SDS-PAGE
2. 2DE-DIGE 2. HPLC
3. HPLC 3. Mass spectroscopy (MS)
4. Mass spectroscopy (MS) 4. Molecular modeling tools(bioinformatics)5. Proteomic modeling tools
(bioinformatics)
8 Proteomics 119
should be compatible with the sample. Someproteomic methods include High-performanceliquid chromatography (HPLC), Mass Spec-trometry, SDS-PAGE, two-dimensional gelelectrophoresis, and in silico protein modeling.There are many sample type, sample preparation,and analytical technology combinations whichcan be used for study in proteomics. Table 8.1compares the broad-based approach with tradi-tional focused approach in proteomics.
8.1.3 Human Proteome Organization
Human Proteome Organization (HUPO) is aninternational scientific organization representingand promoting proteomics through internationalcooperation and collaborations by fostering thedevelopment of new technologies, techniques,and training. It is an international consortium ofnational proteomics research associations, gov-ernment researchers, academic institutions, andindustry partners. HUPO, founded in June 2001,promotes the development and awarenessof proteomics research, advocates proteo-mics researchers throughout the world, andfacilitates scientific collaborations betweenHUPO members and initiatives, organized togain better and complete understanding of thehuman proteome. The Human Proteome Orga-nization is currently working on establishing adefined standard for data submission and anno-tation for the many different proteomics tech-niques currently used to identify and annotateproteins. The proteomics standards initiative(PSI) is a working group of HUPO. It aims todefine data standards for proteomics in order tofacilitate data comparison, exchange, and veri-fication. PSI focuses on the following:• Minimum Information about a Proteomics
Experiment (MIAPE) defines the metadatathat should be provided along with a proteo-mics experiment.
• Data Markup Languages for encoding thedata and metadata.
• Ontologies for consistent annotation andrepresentation.
8.2 Classification of Proteomics
Proteomics is mainly concerned with determin-ing the structure, expression, localization, bio-chemical activity, interactions, and cellular rolesof proteins. According to Graves and Haystead(2002) proteomics can be broadly classified intothree types:1. Structural proteomics—It is in-depth large-
scale analysis of protein structure. It deals withdetermination of the 3D-structure of proteincomplexes or the proteins present in a specificcellular organelle. Protein structure compari-sons can help to identify the functions of newlydiscovered genes. Structural analysis can alsoshow where drugs bind to proteins and whereproteins interact with each other. This isachieved using technologies such asX-ray crystallography and NMR spectroscopy.
2. Expression proteomics—Large-scale analy-sis of expression and differential expressionof proteins is called as expression proteo-mics. This can help to identify the mainproteins found in a particular sample andproteins differentially expressed in relatedsamples, such as diseased versus healthy tis-sue. A protein found only in a diseasedsample may represent a useful drug target ordiagnostic marker. Proteins with similarexpression profiles may also be functionallyrelated. Technologies such as 2D-PAGE andmass spectrometry are used for this.
3. Interaction proteomics—It deals with anal-ysis of interactions between proteins tocharacterize complexes and determine func-tion. The characterization of protein–proteininteractions helps to determine protein
120 I. Ravi
functions and can also show how proteinsassemble in larger complexes. Technologiessuch as affinity purification, mass spectrom-etry, and the yeast two-hybrid system areparticularly useful for this.
8.3 Basic Steps in Proteomics
The following steps are involved in a proteomicsexperiment (Fig. 8.3):1. protein isolation from a biological sample
(e.g., a cell extract) following some experi-mental treatment.
2. fractionation of the resulting proteins (orpeptides, the products of proteome digestion)by methods such as two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE)or liquid chromatography (LC).
3. protein or peptide detection by MS.
4. protein identification through manual inter-pretation or database correlation of massspectra.
8.3.1 Protein Sample Preparation
With technological advances in proteomics,procedures for preparation of protein samplesprior to any particular procedure have alsoadvanced. A number of issues arise in thisrespect, including sample clean-up, fraction-ation, enrichment, and also sample conditionoptimization. This facet of proteomics isbecoming particularly critical in case of high-throughput protocols where the necessary con-ditions of a sample in one stage may directlyconflict with the efficacy of a second stage. Forexample, during the initial step in 2D electro-phoresis and isoelectric focusing, all proteins ina sample are given a net charge of zero; while inthe second step, gel electrophoresis requires anegative charge on all products in the sample inorder to induce movement through the gelmatrix. Many companies, e.g., Millipore, Bio-Rad, Gelifesciences, Invitrogen, Aligent, Beck-mancoulter, Bioproximity, etc., offer pre-packaged kits that allow to prepare samples formany different techniques. They also offer manyprotein samples and other protein technologies.
8.3.2 Determining the Existenceof Proteins in Complex Mixtures
Classically, antibodies to particular proteins orto their modified forms have been used in bio-chemistry and cell biology studies. For morequantitative determinations of protein amounts,techniques such as ELISAs can be used. Forproteomic study, more recent techniques such asMatrix-assisted laser desorption or ionizationhas been employed for rapid determination ofproteins in particular mixtures.
Protein Sample Preparation
Separation of Proteins
(First Dimension Second Dimension)
Protein Fractionation
Protein Detection/Identification(Staining, Imaging, Analysis and extraction)
Mass Spectrophotometric Identification
Protein Sequencing
Bioinformatics/ Data Analysis
Fig. 8.3 Flow diagram of the steps involved inproteomics
8 Proteomics 121
8.3.3 Determining Post-translationally ModifiedProteins
A particular protein can be studied by developingan antibody which is specific to that modifica-tion. For example, there are antibodies whichonly recognize certain proteins when they aretyrosine-phosphorylated, also, there are antibod-ies specific to other modifications. These can thenbe used to determine the set of proteins that haveundergone the modification of interest. For sugarmodifications, such as glycosylation of proteins,certain lectins have been discovered which bindsugars. A more common way to determine post-translational modification of interest is by 2D gelelectrophoresis. Recently, another approachcalled PROTOMAP has been developed whichcombines SDS-PAGE with shotgun proteo-mics to enable detection of changes in gel-migration such as those caused by proteolysis orpost-translational modification.
8.4 Techniques Used in Proteomics
Some of the techniques used in proteomics studyare given below:1. One- and two-dimensional gel electropho-
resis is used to identify the relative mass of aprotein and its isoelectric point.
2. HPLC is used for separation of proteins.3. X-ray crystallography and nuclear mag-
netic resonance are used to characterize the3D structure of peptides and proteins. How-ever, low-resolution techniques such as cir-cular dichroism, Fourier transform infraredspectroscopy, and small angle X-ray scatter-ing can be used to study the secondarystructure of proteins.
4. Tandem mass spectrometry (MS/MS)combined with reverse phase chromatogra-phy or 2D electrophoresis is used to identify(by de novo peptide sequencing) and quantifyall the levels of proteins found in cells.
5. Mass spectrometry, often MALDI-TOF, isused to identify proteins by peptide massfingerprinting (PMF). Less commonly thisapproach is used with chromatography and/orhigh-resolution mass spectrometry.
6. Affinity chromatography, Yeast two-hybrid techniques, Fluorescence resonanceenergy transfer (FRET), and Surfaceplasmon resonance (SPR) are used to iden-tify protein–protein and protein-DNA bind-ing reactions.
7. X-ray tomography is used to determine thelocation of labeled proteins or protein com-plexes in an intact cell. It is frequently cor-related with images of cells from light-basedmicroscopes.
8. Software-based image analysis is utilized toautomate the quantification and detection ofspots within and among gel samples.Some of these techniques have been dis-
cussed in detail.
8.4.1 One- and Two-Dimensional GelElectrophoresis
One- and two-dimensional gel electrophoresis isused to identify the relative mass and isoelectricpoint of a protein.
8.4.1.1 ElectrophoresisThe migration of charged colloidal particles ormolecules through a solution under the influenceof an applied electric field is usually provided byimmersed electrodes. It can also be described asa method of separating substances, especiallyproteins and analyzing molecular structure basedon the rate of movement of each component in acolloidal suspension under the influence of anelectric field. An analyte is a chemical substancethat is the subject of chemical analysis. Separa-tion by electrophoresis depends on differences inthe migration velocity of ions or solutes througha given medium in an electric field. The elec-trophoretic migration velocity of an analyte is
122 I. Ravi
vp ¼ lpE where E is the electric field strengthand lp is the electrophoretic mobility.
The electrophoretic mobility is inverselyproportional to frictional forces in the buffer,and directly proportional to the ionic charge ofthe analyte. The forces of friction against ananalyte are dependent on the analyte’s size andthe viscosity (g) of the medium. Analytes withdifferent frictional forces or different chargeswill separate from one another when they movethrough a buffer. At a given pH, the electro-phoretic mobility of an analyte is:
lp ¼z
6pr
where, r is the radius of the analyte and z is thenet charge of the analyte.
Differences in the charge to size ratio ofanalyte cause differences in electrophoreticmobility. Small, highly charged analytes havegreater mobility, whereas large, less chargedanalytes have lower mobility. Electrophoreticmobility is an indication of an analyte’s migra-tion velocity in a given medium. The net forceacting on an analyte is the balance of two forces:the electrical force acting in favor of motion, andthe frictional force acting against motion. Thetwo forces remain steady during electrophoresis,thus the electrophoretic mobility is a constant fora given analyte under a given set of conditions.
Electrophoresis has a wide variety of appli-cations in proteomics, forensics, molecular biol-ogy, genetics, biochemistry, and microbiology.One of the most common uses of electrophoresisis to analyze differential expression of genes.Healthy and diseased cells can be identified bydifferences in the electrophoretic patterns oftheir proteins. Proteins can also be characterizedsimilarly, and some information about theirstructure can be derived from the masses offragments in the gel.
8.4.1.2 Two-Dimentional GelElectrophoresis
Two-dimensional polyacrylamide gel electro-phoresis (2DE) was first described by O’Farrell
in 1975 and has evolved markedly as oneof the core technologies for the analysis ofcomplex protein mixtures extracted from bio-logical samples since then. The proteins areseparated in 2 steps according to 2 independentproperties [isoelectric point (pI) and molecularweight (MW)].
The proteins are made up of amino acidswhich may have positive, negative, or no char-ges. In addition, amino acids can be hydrophobicor hydrophilic. These amino acid properties arecombined in the molecule to determinethe electrostatic and amphiphilic properties ofthe proteins. When a protein of charge q isplaced in an electric field, E, it experiences anelectrical force given by: F = q 9 E. Under theinfluence of this force, the protein moves untilthe force becomes zero (F = 0). This principleis used to make proteins move in a liquid orsolid media. A common medium used is Poly-acrylamide (PAA). PAA is a flexible, elasticpolymer that allows particles to move inverselyproportional to their sizes; i.e., smaller particlesmove faster than larger particles. Thus a mixtureof proteins in a PAA gel under the influence of aforce F will separate in individual moleculesdepending on their sizes and charges. Two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) is a form of gel electrophoresis inwhich proteins are separated and identified intwo dimensions oriented at right angles to eachother. Small changes in charge and mass caneasily be detected by this method, because it israre that two different proteins will resolve to thesame place in both dimensions. A 2D gel canresolve one thousand to two thousand proteins,which appear, after staining, as dots in the gel.
The three main advantages of this techniqueare its robustness, its parallelism, and its uniqueability to analyze complete proteins at highresolution. This technique is useful when com-paring two similar samples to find specific pro-tein differences. Using 2D-PAGE, hundreds tothousands of polypeptides can be analyzed in asingle run. The proteins can be separated in pureform from the resultant spots which can bequantified and further analyzed by mass
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spectrometry, depending on their resolution.Polypeptides can also be probed with antibodiesand tested for post-translational modifications.2D-PAGE is also used to study differentialexpression of proteins between cell types. Thetwo main drawbacks are its very low efficiencyin the analysis of hydrophobic proteins, and highsensitivity toward the dynamic range and quan-titative distribution issues. It requires a largeamount of sample handling, limited reproduc-ibility, and a smaller dynamic range than otherseparation methods. It is also not automated forhigh-throughput analysis. Certain proteins aredifficult for 2D-PAGE to separate such as thosethat are in low abundance, acidic, basic, hydro-phobic, very large, or very small.
8.4.2 Centrifugation
Centrifugation is one of the most important andwidely applied research techniques in biochem-istry, cellular and molecular biology, and inmedicine. In proteomics it plays a vital role inthe fundamental and necessary process of iso-lating proteins. This process begins with intactcells or tissues. Before the proteins can beobtained, the cells must be broken open byprocesses such as snap freezing, sonication,homogenization by high pressure, or grindingwith liquid nitrogen. Once the cells have beenopened up, all of their contents including cellmembranes, RNA, DNA, and organelles will bemixed in the solvent with the proteins. Centri-fugation is used for separating out all the non-protein material. Within the centrifuge samplesare spun at high speeds and the resulting forcecauses particles to separate based on their den-sity. Centrifugation is also used for removingcells or other suspended particles from theirsurroundings, isolating viruses and macromole-cules, including DNA, RNA, proteins, and lipidsor establishing physical parameters of theseparticles from their observed behavior duringcentrifugation, separating from dispersed tissuethe various subcellular organelles includingnuclei, mitochondria, chloroplasts, golgi bodies,
lysosomes, peroxisomes, glyoxysomes, plasmamembranes, endoplasmic reticulum, polysomes,and ribosomal subunits. Once the mixture ofproteins has been isolated using centrifugation,one of several methods to separate out individualproteins can be used for further study.
8.4.3 Protein Separation-Chromatography
To obtain a pure protein sample, a protein has tobe isolated from all other proteins and cellularcomponents. This can prove to be difficultbecause a single protein often makes up only1 % of the total protein concentration of a cell.Therefore, 99 % of the protein components of asample must be removed before it can be clas-sified as pure. Protein separations can be done bychromatography. There are several properties ofproteins that can be used to separate them. Dif-ferent types of chromatography take advantageof different properties. Proteins can be separatedon the basis of size, shape, hydrophobicity,affinity to molecules or charge. All methodsutilize an insoluble stationary phase and amobile phase that passes over it. The mobilephase is commonly a liquid solution whichcontains the protein that has to be isolated. Thestationary phase on the other hand is made up ofa group of beads, usually based on a carbohy-drate or acrylamide derivative, that are bound toionically charged species, hydrophobic charac-ters, or affinity ligands.
In column chromatography, when a proteinsample is applied to the column, it equilibratesbetween the stationary phase and the mobilephase. Depending on the type of chromatogra-phy, proteins with certain characteristics willbind to the stationary phase while those lackingthe sought characteristics will remain in themobile phase and pass through the column. Forexample in ion exchange chromatography, apositively charged protein binds to a negativelycharged stationary phase, while the negativelycharge protein will be eluted from the columnwith the mobile phase. The final step involves
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displacing the protein from the stationary phase,also known as elution. This is done by intro-ducing a particle which will compete with theprotein binding site on the stationary phase.Various commercial columns are available; spe-cifically Bio-Rad, Sigma-Aldrich, GE Healthcare,etc., offer a variety of chromatography columns(Fig. 8.4).
8.4.4 Emerging and MiscellaneousTechnologies in Proteomics
8.4.4.1 Multi-dimentional ProteinIdentification Technologyor Orthogonal Separations
Multi-dimentional protein identification technol-ogy (MudPIT) is a chromatography-based pro-teomic technique in which a complex peptidemixture is prepared from a protein sample andloaded directly onto a triphasic microcapillarycolumn packed with reversed phase, strong cat-ion exchange, and reversed phase HPLC gradematerials. Once the complex peptide mixture isloaded the column is placed directly in-line with atandem mass spectrometry (MS/MS). The MS/MS data generated from a MudPIT run is thensearched to determine the protein content of theoriginal sample.
MudPIT is a robust and widely acceptedmethod for protein identification from a widevariety of samples. It is an excellent tool for bothqualitative and quantitative proteomic analyses.
Through on-line 2D HPLC, complex peptidesmixtures can be well separated. For a relativelysimple sample, better results are obtained fromMudPIT, compared to single dimension liquidchromatography method. It was developed as amethod to analyze the highly complex samplesnecessary for large-scale proteome analysis byelectrospray ionization, MS/MS, and databasesearching. This method couples a 2D liquidchromatography separation of peptides on a mi-crocapillary column with detection in a tandemmass spectrometer. In the MudPIT, a protein ormixture of proteins is first reduced (to breakcysteine disulfide bonds), alkylated (to preventreformation of disulfide bonds), and digested intoa complex mixture of peptides.
MudPIT has been used in a wide range ofproteomics experiments, including large-scalecatalogs of proteins in cells and organisms,profiling of organelle and membrane proteins,identification of protein complexes, determina-tion of post-translational modifications andquantitative analysis of protein expression. InMudPIT biochemical fractions containing manyproteins are directly proteolyzed and the enor-mous number of peptides generated, are sepa-rated by 2D liquid chromatography beforeentering the mass spectrometer. Instead ofMALDI-TOF, MS/MS is employed so that, afterthe mass of a peptide is measured, the peptide isfragmented using a collision-induced dissocia-tion cell, and the masses of the fragmentationproducts are determined.
Fig. 8.4 Chromatogram showing separation based onsignals given by a detector. X axis: Time in Min orvolume in ml. Y axis: Signal. tm — the time required for
the mobile phase to travel the entire length of the column,tr—the time required for a specific protein to elute fromthe column
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8.4.4.2 Isotope-Coded Affinity TagIsotope-coded affinity tags (ICATs) are gel-freemethod for quantitative proteomics that relieson chemical labeling reagents. These chemicalprobes consist of three general elements: a reac-tive group capable of labeling a defined aminoacid side chain (e.g., iodoacetamide to mod-ify cysteine residues), an isotopically coded lin-ker and a tag (e.g., biotin) for the affinity isolationof labeled proteins/peptides. For the quantitativecomparison of two proteomes, one sample islabeled with the isotopically light (d0) probe andthe other with the isotopically heavy (d8) version(Fig. 8.5). To minimize error, both samples arethen combined, digested with a protease (i.e.,trypsin), and subjected to avidin affinity chro-matography to isolate peptides labeled with iso-tope-coded tagging reagents. These peptides arethen analyzed by liquid chromatography–massspectrometry (LC–MS). The ratios of signalintensities of differentially mass-tagged peptidepairs are quantified to determine the relativelevels of proteins in the two samples. The originaltags were developed using deuterium, but latertags using 13C were used instead to circumventissues of peak separation during liquid chroma-tography due to the interaction of deuterium withthe stationary phase of the column.
For samples that are not amenable to meta-bolic labeling, such as when analyzing clinicalsamples (e.g., biological fluids, tissue samples)or when experimental time is limited, chemical
or enzymatic stable isotopic labeling methodsare available for quantitative proteomic analy-ses. These include strategies to add isotopicatoms or isotope-coded tags to peptides or pro-teins. A rapid and relatively inexpensive methodof chemical labeling is stable isotope dimethy-lation which uses formaldehyde in deuteratedwater to label primary amines with deuteratedmethyl groups. This approach also does notchange the ionic state of the labeled peptidesbecause of the reductive amination that occurs,so their chemical properties remain the same asthose of unlabeled peptides. Benefit of thisapproach is that many samples are amenable toformaldehyde fixation, which is fast and cheapcompared to other labeling reagents. Thisrequires using pure samples or sample prepara-tion to reduce the complexity of biologicalsamples to minimize the number of peaksdetected by MS.
Protein labeling with ICAT followed by MS/MS allows sequence identification and accuratequantification of proteins in complex mixtures,and has been applied to the analysis of globalprotein expression changes, protein changes insubcellular fractions, components of proteincomplexes, protein secretion, and body fluids.
8.4.4.3 Label-Free TagsLabel-free methods for both relative and abso-lute quantitation have been developed as a rapid
Linker region (heavy or light) Biotin
Sample 1 peptides ICAT (light) Light Peptides
ICAT-tagged peptide Detector (LC-MS)Purification
Sample 2 peptides ICAT (heavy) Heavy peptides
Fig. 8.5 Process of ICAT used in proteomics
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and low-cost alternative to other quantitativeproteomic approaches. These strategies are idealfor large-sample analyses in clinical screening orbiomarker discovery experiments but are lessreliable for measuring small changes. Unlikeother quantitation methods, label-free samplesare separately collected, prepared, and analyzedby liquid chromatography–mass spectrometry,LC–MS, or LC–MS/MS. Hence, label-freequantitation experiments need to be more care-fully controlled than stable isotope methods toaccount for any experimental variations. Proteinquantitation is performed using either ion peakintensity or spectral counting.
Relative quantitation by ion peak intensityrelies on LC–MS only. The direct MS m/z valuesfor all ions are detected and their signal intensi-ties at a particular time recorded. The signalintensity from electrospray ionization has beenreported to highly correlate with ion concentra-tion, therefore the relative peptide levels betweensamples can be determined directly from thesepeak intensities. Because of the large amount ofdata collected from these experiments, sensitivecomputer algorithms are required for automatedion peak alignment and comparison. Label-freerelative quantitation by spectral counts entailscomparing the sum of the MS/MS spectra from agiven peptide across multiple samples, which hasbeen shown to directly correlate with proteinabundance. Besides relative quantitation, label-free methods can be used to determine theabsolute concentration of proteins in a sample.One method entails determining the exponen-tially modified protein abundance index (em-PAI), which estimates protein abundance basedon the number of peptides detected and thenumber of theoretically observed tryptic peptidesfor each protein, is used to determine theapproximate absolute protein abundance inlarge-scale proteomic analyses. Another method,absolute protein expression (APEX) is based onspectral counts and uses correction factors tomake protein abundance proportional to thenumber of peptides observed.
8.4.4.4 Flourescence Resonance EnergyTransfer
Flourescence resonance energy transfer (FRET)is an important tool to study protein–proteininteractions, protein-DNA interactions, proteinconformational changes and other moleculardynamics quantification. It is used to detectprotein binding, providing spatial and temporalinformation about the interaction. It is a type ofFluorescence Spectroscopy using two fluores-cent dyes with overlapping emission andabsorption spectra, which is used to indicateproximity of labeled molecules. This techniqueis useful for studying interactions of moleculesand protein folding. It uses fluorescent energytransfer to visualize protein interactions. Fluo-rophores are fused to the proteins of interest andthen bombarded with light at the excitationwavelength. The first fluorophore transfers someof the energy it absorbs from the light source tothe second fluorophore, which in turn emitssome of its energy into the environment where itis visible with the use of a fluorescentmicroscope.
Since the discovery of FRET by TheodorForster in 1948, it has become useful tool inbiology for three reasons: (1) FRET is reallysensitive in the range of 100 Å and below, thescale at which transactions between biologicalmacromolecules and complexes occur; (2) theinstrumentation is extremely sensitive and isreadily amenable to miniaturization, highthroughput, and automation; and (3) cell per-meable and genetically encodable FRET probesenable the real-time quantitation of dynamiccellular processes in live cells.
8.4.4.5 Mass SpectrometryMass spectrometry (MS) is an importantemerging method for the characterization ofproteins. MS is a technique in which gas phasemolecules are ionized and their mass-to-chargeratio is measured by observing acceleration
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differences of ions when an electric field isapplied (Fig. 8.6). Lighter ions will acceleratefaster and be detected first. If the mass is mea-sured with precision then the composition of themolecule can be identified. In the case of pro-teins, the sequence can be identified. Mostsamples submitted to MS are a mixture ofcompounds. A spectrum is acquired to give themass-to-charge ratio of all compounds in thesample. MS throws light on molecular mecha-nisms within cellular systems. It is used foridentifying proteins, functional interactions, andit further allows for determination of subunits.Several configurations of mass spectrometersthat combine electrospray ionization (ESI) andmatrix-assisted laser desorption ionization(MALDI) with a variety of mass analyzers(linear quadrupole mass filter [Q], time-of-flight[ToF], quadrupole ion trap, and Fourier trans-form ion cyclotron resonance [FTICR] instru-ment) are routinely used (Yates et al. 2009).
Whole protein mass analysis is primarilyconducted using either TOF, MS, or FTICR.These two instruments are preferable because oftheir wide mass range and in the case of FTICR,its high-mass accuracy. Mass analysis of prote-olytic peptides is a much more popular methodof protein characterization, as cheaper instru-ment designs can be used for characterization.Additionally, sample preparation is easier oncewhole proteins have been digested into smallerpeptide fragments. The most widely usedinstrument for peptide mass analysis are theMALDI time-of-flight instruments as they per-mit the acquisition of peptide mass finger-prints (PMFs) at high pace. Multiple stagequadrupole-time-of-flight and the quadrupoleion trap also find use in this application.
The relative abundance of an ion can also bemeasured using MS. Different compounds havedifferential ionization capabilities, thereforeintensity of an ion is not in a direct correlation toconcentration. It is an analytical method whichhas a variety of uses outside of proteomics, suchas isotope and dating, trace gas analysis, atomiclocation mapping, pollutant detection, and spaceexploration. This technique was discoveredduring studies of gas excitation in a chargedenvironment, more than 100 years agoby J. J. Thomson in 1913.
The ionization methods used for the majorityof biochemical analyses are:1. ESI
ESI is one of the atmospheric pressure ioni-zation (API) techniques and is well suited to theanalysis of polar molecules ranging from lessthan 100 Da to more than 1,000,000 Da inmolecular mass. In ESI the ions of interest areformed from solution by applying a high electricfield to the tip of a capillary, from which thesolution will pass through. The sample will besprayed into the electric field along with a flowof nitrogen to promote desolvation. Droplets willform and will evaporate in a vacuumed area.This causes an increase in charge on the dropletsand the ions are now said to be multiply charged.These multiply charged ions can then enter theanalyzer (Andersen et al. 1996). ESI is a methodof choice because of the following properties:(1) The ‘‘softness’’ of the phase conversionprocess allows very fragile molecules to beionized intact and even in some non-covalentinteractions to be preserved for MS analysis. (2)The eluting fractions through liquid chroma-tography can then be sprayed into the massspectrometer, allowing for the further analysis of
Data Analysis
Sample
Ion Source Mass Analyzer Detector
Fig. 8.6 Schematicdiagram of a massspectrometer
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mixtures. (3) The production of multiplycharged ions allow for the measurement of high-mass biopolymers. Multiple charges on themolecule will reduce its mass to charge ratiowhen compared to a single charged molecule.Multiple charges on a molecule also allow forimproved fragmentation which in turn allows fora better determination of structure.2. MALDI
MALDI (Hillenkamp et al. 1991) deals withthermolabile, non-volatile organic compoundsespecially those of high molecular mass and isused successfully in biochemical areas for theanalysis of proteins, peptides, glycoproteins,oligosaccharides, and oligonucleotides. It isrelatively straightforward to use and reasonablytolerant to buffers and other additives. The massaccuracy depends on the type and performanceof the analyzer of the mass spectrometer, butmost modern instruments are capable of mea-suring masses to within 0.01 % of the molecularmass of the sample, at least up to ca. 40,000 Da.In MALDI, the molecular ions of interest areformed by pulses of laser light impacting on thesample isolated within an excess of matrixmolecules. This enables the determination ofmasses of large biomolecules and syntheticpolymers greater than 200,000 Daltons withoutdegradation of the molecule of interest. Theadvantages of MALDI are its robustness, highspeed, and relative immunity to contaminantsand biochemical buffers. A type of mass
spectrometer often used with MALDI is TOF orTime-of-Flight mass spectrometry. This enablesfast and accurate molar mass determinationalong with sequencing repeated units and rec-ognizing polymer additives and impurities. Thistechnique is based on an ultraviolet absorbingmatrix where the matrix and polymer are mixedtogether along with excess matrix and a solventto prevent aggregation of the polymer. Thismixture is then placed on the tip of a probe; thenthe solvent is removed while under vacuumconditions. This creates co-crystallized polymermolecules that are dispersed homogeneouslywithin the matrix. A pulsing laser beam is set toan appropriate frequency and energy is shot tothe matrix, which becomes partially vaporized.As a result the homogeneously dispersed poly-mer within the matrix is carried into the vaporphase and becomes charged.
Finally, in the TOF analyzer the moleculesfrom a sample are imparted identical transla-tional kinetic energies because of the electricalpotential energy difference. These ionic mole-cules travel down an evacuated tube with noelectrical field and of the same distance. Thesmallest ions arrive first at the detector, whichproduces a signal for each ion (Fig. 8.7). Thecumulative data from multiple laser shots yield aTOF mass spectrum, which translates thedetector signal into a function of time, which canbe used to calculate the mass of the ion. Theseanalyzers measure the mass/charge ratio of
ion detector
Accelaration zone
light ions
Flight path
Ionisation zone
Sample
Heavy ions
Measurement of timeVacuum Chamber
Fig. 8.7 MALDI-TOFmass spectrometry
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intact ionized biomolecules, as well as theirfragmentation spectra, with high accuracy andhigh speed.3. Surface-enhanced laser desorption ioniza-
tion (SELDI)–ToF MSThis is a technique for the enrichment of
proteins with specific chemical characteristicsand combines chromatography with MS. Proteinchip arrays have been designed that containchemically or biochemically treated surfaces.The sample, consisting of crude extracts ormixtures of whole proteins, is applied to thesurface. After a series of washes, the targetedproteins are selectively retained. An energy-absorbing solution is added to the surface andthe sample is subjected to laser desorption ion-ization (Fig. 8.8). The formed ions are measuredusing a ToF mass analyzer as previouslydescribed. Characteristic features of spectra canbe used for prognostic purposes.
The technique has at its heart a means ofenriching for proteins with certain chemicalcharacteristics that determine their interactionwith a set of specific surfaces used as a laserdesorption ionization target. One advantage ofthis chip-based technique is that crude samplescan readily and rapidly be analyzed with highthroughput. The key disadvantage is that themass spectrum obtained does not enable identi-fication of the proteins analyzed and furtherwork is therefore required. The data obtained areanalyzed to recognize critical features that maybe used as markers for diagnosis or prognosis.For example, analysis of serum of ovarian can-cer by an iterative algorithm that identified aproteomic pattern to discriminate healthypatients from those with malignant disease,using the minimum amount of information. Astudy on prostate cancer and lung cancer reveals
the general applicability of this technique. Fur-thermore, there are indications that determina-tion of a serum protein level (e.g., high-densitylipoproteins) can be monitored effectively withSELDI-ToF and therefore, indicators of hema-tologic disease could theoretically be mea-sured. Recently, study of non-small-cell lungtumors showed the potential of this technique forthe detection of disease markers.4. MS/MS
MS/MS is used to produce structural infor-mation about a compound by fragmenting spe-cific sample ions inside the mass spectrometerand identifying the resulting fragment ions togenerate structural information regarding theintact molecule. It also enables specific com-pounds to be detected in complex mixtures onaccount of their specific and characteristicfragmentation patterns.
MS/MS are instruments that have more thanone analyzer and so can be used for structuraland sequencing studies. The two analyzers areseparated by a collision cell into which an inertgas (e.g., argon, xenon) is admitted to collidewith the selected sample ions and bring abouttheir fragmentation. The analyzers can be of thesame or of different types, the most commoncombinations being e.g., quadrupole–quadrupole,magnetic sector-quadrupole, magnetic sector-magnetic sector, quadrupole-time-of-flight.Fragmentation experiments can also be per-formed on certain single analyser mass spec-trometers such as ion trap and time-of-flightinstruments.
The most common usage of MS/MS in bio-chemical areas is the product or daughter ionscanning experiment which is particularly suc-cessful for peptide and nucleotide sequencing. Itinvolves more than one step of mass selection or
Hydrophobic Ion source Mirror Laser
Chemical Surfaces
Ionic Field free regionWashing Detector
DNA
Biochemical Surfaces
Antibody On chip sample fractionation
Fig. 8.8 The technique ofSELDI-ToF MS
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analysis, and fragmentation is usually inducedbetween the steps. One example of an applica-tion of tandem mass spectrometry is proteinidentification. The first mass analyzer isolatesions of a particular m/z value that represent asingle species of peptide among many intro-duced into and then emerges from the ionsource. Those ions are then accelerated into acollision cell containing an inert gas such asargon to induce fragmentation. This process isdesignated collisionally induced dissociation(CID) or collisionally activated dissociation(CAD). The m/z values of the fragment ions arethen measured in a second mass analyzer toproduce amino acid sequence information.
Uses of MSMass spectrometry can be used to identify
proteins through various mass spectrometrytechniques and also in protein quantification.
Top-down proteomics is a method of pro-tein identification that uses an ion trapping massspectrometer to store an isolated protein ion formass measurement and tandem mass spectrom-etry analysis. Recently, a top-down approachhas been developed for mapping the connectiv-ity of the disulfide-rich peptide hedyotide B2.This method allows rapid characterization of thedisulfide pattern of cystine-knot miniproteinssuch as cyclotides, conotoxin, knottin, and plantdefensins. In this approach the complete proteinsare directly analyzed by using mass spectrome-ter without solution digestion. The advantages ofthe top-down approach are that it can sometimeprovide the complete coverage of the protein.But since whole proteins are hard to handlebiochemically compared to small peptide pieces,it makes top-down approach difficult to analyze.
Bottom-up proteomics is a common methodto identify proteins and characterize their aminoacid sequences and post-translational modifica-tions by proteolytic digestion of proteins prior toanalysis by mass spectrometry. The proteins mayfirst be purified by a method such as gel elec-trophoresis resulting in one or a few proteins ineach proteolytic digest. Alternatively, the crudeprotein extract is digested directly, followed byone or more dimensions of separation of thepeptides by liquid chromatography coupled to
mass spectrometry, a technique known as shot-gun proteomics. By comparing the masses of theproteolytic peptides or their tandem mass spec-tra with those predicted from a sequence data-base or annotated peptide spectral in a peptidespectral library, peptides can be identified andmultiple peptide identifications assembled intoprotein identification.
Another use of mass spectrometry in proteo-mics is protein quantification. By labeling pro-teins with stable heavier isotopes the relativeabundance of proteins can be determined.Commercial kits such as iTRAQ (Applied Bio-systems) are available for this to obtain a high-throughput level. One of the most powerful waysto identify a biological molecule is to determineits molecular mass together with the masses ofits component building blocks after fragmenta-tion. In silico proteome analysis can also bedone to facilitate proteomics experiments usingMS (Cagney et al. 2003).
8.4.4.6 Peptide Mass FingerprintingThis technique was developed in 1993 inde-pendently by many groups. Protein identificationby MS can be performed using sequence-spe-cific peptide fragmentation or PMF, also knownas peptide mass mapping. The standard approachto identify proteins includes separation of pro-teins by gel electrophoresis or liquid chroma-tography. Subsequently, the proteins are cleavedwith sequence specific endoproteases, generallytrypsin. Following digestion, the generatedpeptides are investigated by determination ofmolecular masses or generation of peptidefragments. For protein identification, the exper-imentally obtained masses are compared withthe theoretical peptide masses of proteins storedin databases. MALDI-MS is the most commonlyused technique to perform PMF. The predomi-nant detection of singly charged peptide mole-cules by MALDI-MS facilitates the evaluationof PMFs significantly. Although PMF is aneffective tool for the identification of relativelypure proteins, it often fails to identify proteinmixtures. Separation of complex protein samplesby high-resolution 2D gel electrophoresis (2DE)
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is well adapted to protein identification withPMF. On the other hand, the application of PMFin combination with one-dimensional gel elec-trophoresis or liquid chromatography must beadjusted to the separation capacity. A frequentlyused strategy to identify proteins by MS is tofirst generate a PMF because of the simplicity ofthe method. Peptide fragments can be generateddirectly by MALDI-MS using post-source decay(PSD)-MALDI-MS or collision-induced disso-ciation (CID)-MALDI-MS/MS. The relativenew MALDI-ion traps, MALDI-Q-TOF, andMALDI-TOF/TOF instruments are particularlysuitable for this purpose. Alternatively, nano-electrospray ionization tandem mass spectrom-etry (nano-ESI-MS/MS) is used frequently toachieve sequence information. Furthermore, thepower and limitations of PMF are illustrated bythe identification of phosphorylation sites,splicing variants, proteins with disulfide bridges,large proteins, and protein fragments. Anotherfocus is the MS-Screener software which can beused for the advanced evaluation of PMFs.
The advantage of this method is that only themasses of the peptides have to be known. Time-consuming de novo peptide sequencing is thenunnecessary. A disadvantage is that the proteinsequence has to be present in the database ofinterest. Additionally most PMF algorithmsassume that the peptides come from a singleprotein.
8.4.4.7 Shot Gun ProteomicsShotgun proteomics refers to the use of bottom-up proteomics techniques in identifying pro-teins in complex mixtures using a combinationof high-performance liquid chromatogra-phy combined with mass spectrometry. In thismethod, the proteins in the mixture are digeste-d and the resulting peptides are separated byliquid chromatography. Tandem mass spec-trometry is then used to identify the peptides.The advantage of Shotgun proteomics allowsglobal protein identification as well as the abilityto systematically profile dynamic proteomes. Italso avoids the modest separation efficiency andpoor mass spectral sensitivity associated with
intact protein analysis. The disadvantage isdynamic exclusion filtering that is often used inshotgun proteomics maximizes the number ofidentified proteins at the expense of randomsampling. This problem may be exacerbated bythe undersampling inherent in shotgunproteomics.
8.4.4.8 Nuclear Magnetic ResonanceNuclear magnetic resonance (NMR) is a physi-cal phenomenon in which nuclei in a magneticfield absorb and re-emit electromagnetic radia-tion. This energy is at a specific resonance fre-quency which depends on the strength of themagnetic field and the magnetic properties ofthe isotope of the atoms.
The principle of NMR usually involves twosequential steps:• The alignment (polarization) of the magnetic
nuclear spins in an applied, constant magneticfield.
• The perturbation of this alignment of thenuclear spins by employing an electro-mag-netic, usually radio frequency pulse. Therequired perturbing frequency is dependentupon the static magnetic field and the nucleiof observation.The two fields are usually chosen to be per-
pendicular to each other as this maximizes theNMR signal strength. The resulting response bythe total magnetization of the nuclear spins is thephenomenon that is exploited in NMR spec-troscopy and magnetic resonance imaging. Bothuse intense applied magnetic fields in order toachieve dispersion and very high stability todeliver spectral resolution. NMR structuredetermination is limited currently by size con-straints and lengthy data collection and analysistimes (often months), and the method is bestapplied to proteins smaller than 250 aminoacids. On the other hand, NMR experiments donot require crystals and samples appropriate forstructure determination can be identified withinminutes of protein purification.
Structural proteomics elucidates structure–function relationships of uncharacterized geneproducts based on the 3D protein structure. It
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proposes biochemical and cellular functions ofunannotated proteins and thereby identifiespotential drug design and protein engineeringtargets. NMR-based structural proteomics toge-ther with X-ray crystallography provides acomprehensive structural database to predict thebasic biological functions of hypothetical pro-teins identified by the genome projects.
8.4.4.9 X-ray CrystallographyIn order to determine the function of proteins thedetermination of the 3D structure of proteins isrequired. The X-ray crystallography is a majortool to explore the structure–function of macro-molecule. X-ray crystallography currently isperceived as the potential workhorse for struc-tural proteomics, because if provided with awell-diffracting crystal it is possible to determinea 3D structure in hours. The three components inan X-ray crystallographic analysis are a proteincrystal, a source of X-rays, and a detector. X-raycrystallography is used to investigate molecularstructures through the growth of solid crystals ofthe molecules they study. Crystallographers aimhigh-powered X-rays at a tiny crystal containingtrillions of identical molecules. The crystalscatters the X-rays onto an electronic detector.After each blast of X-rays, lasting from a fewseconds to several hours, the crystal is rotated byentering its desired orientation into the computerthat controls the X-ray apparatus. This enablesthe scientists to capture in three dimensions howthe crystal scatters, or diffracts, X-rays. Theintensity of each diffracted ray is fed into acomputer, which uses a mathematical equation tocalculate the position of every atom in the crys-tallized molecule. The result is a 3D digitalimage of the molecule.
Crystallographers measure the distancesbetween atoms in angstroms. The X-rays usedby crystallographers are approximately0.5–1.5 Å long. The scattering of X-rays is alsoknown as ‘‘X-ray diffraction’’. Such scattering isthe result of the interaction of electric andmagnetic fields of the radiation with the elec-trons in the atoms of the crystal.
The patterns are a result of interferencebetween the defracted X-rays governed byBragg’s Law: 2d sin h = n * k, where d is thedistance between two regions of electron den-sity, h is the angle of defraction, k is the wave-length of the defracted X-ray, and n is aninteger. If the angle of reflection satisfies thefollowing condition:
sin h ¼ ðn � kÞ2d
the defracted X-rays will interfere construc-tively. Otherwise, destructive interferenceoccurs.
Drawbacks of X-ray crystallography are thatthe sample needs to be in a solid form, thesample must be present in a large enoughquantity to be studied and the sample is oftendestroyed by the X-ray radiation used to study it.No sample in the gas or liquid state can beanalyzed via X-ray crystallography. Also, rare orhard to synthesize samples may be difficult tostudy, because there may not be enough of thesample for the radiation to provide a clearimage. Further, studying biological samples canbe problematic because the radiation used tostudy the samples will harm or destroy the livingtissues. X-ray crystallography also takes a lot oftime to complete one protein structure.
8.4.4.10 X-ray TomographyA new branch of X-ray microscopy is being usedin proteomics analysis, called X-ray tomogra-phy. This method uses projected images to cal-culate and reconstruct a 3D object. Thistechnology is being used in proteomics todetermine the location of labeled proteins orlarge complexes within a cell. This techniquecan also be used in conjunction with images ofcells from light-based microscopes to helpidentify where a protein is located and how thislocation factors into its function and identifica-tion. X-ray tomography is used to determine thelocation of labeled proteins or protein complexesin an intact cell.
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8.4.4.11 Laser Capture MicrodissectionLaser capture microdissection or LCM is aprocess that isolates and removes distinct pop-ulations from a tissue. This facilitates the com-parison of diseased tissue with normal tissuefrom an organism. In LCM, an infrared laserbeam melts a thermosensitive polymer film thattraps a specific group of cells. This polymer filmis then extracted and moved to a test tube wherean extraction buffer is used to remove the groupsof cells for more advanced proteomics analysissuch as 2D-PAGE, Ion Chromatography, etc.This technology will become more useful assystems with higher sensitivity for analysis ofsmaller amounts of tissue are developed andrealized.
8.4.4.12 Proteomic Complex DetectionUsing Sedimentation
Proteomic complex detection using sedimenta-tion (ProCoDeS) is a technique for the high-throughput identification of both soluble andmembrane proteins that are found in stablecomplexes. Relative sizes of protein complexesare estimated via their sedimentation in a gra-dient. In this case, a rate zonal gradient (RZG) isused to estimate the relative size of proteincomplexes. The distribution of a protein ofinterest in this sedimentation can be detectedusing classic techniques such as Western Blot-ting or ICAT. This can be done for a largenumber of proteins. Thus, ProCoDeS can beused to identify stable protein complexes and isespecially well suited for the screening ofunrefined cellular material to help find newproteins that cannot be discovered because theyexist in protein complexes such as proteinsfound in protein membranes.
8.4.4.13 Protein InteractionsMost proteins function in collaboration withother proteins and one goal of proteomics is toidentify which proteins interact. This is espe-cially useful in determining potential partners incell signaling cascades. Several methods are
available to probe protein–protein interactions.The traditional method is yeast two-hybridanalysis. New methods include protein micro-arrays, immunoaffinity chromatography fol-lowed by mass spectrometry, dual polarizationinterferometry, and experimental methods suchas phage display and computational methods.Protein interaction is crucial for every organism.Some proteins are composed of more than onepolypeptide chain, and the interactions betweenthe different peptides are necessary for the wholeprotein to function.
In many experiments and computationalstudies, the focus is on interactions between twodifferent proteins. However, one protein caninteract with other copies of itself (oligomeri-zation), or three or more different proteinsinteracting. The stoichiometry of the interactionis also important—that is, how many of eachprotein involved are present in a given reaction.Some protein interactions are stronger thanothers, because they bind together more tightly.The strength of binding is known as affinity.Proteins will only bind each other spontaneouslyif it is energetically favorable. Energy changesduring binding are another important aspect ofprotein interactions. Many of the computationaltools that predict interactions are based on theenergy of interactions. Predicting the interac-tions can help scientists predict pathways in thecell, potential drugs, and antibiotics and proteinfunctions.
8.4.4.14 Protein chipsProtein chips, also referred to as protein arraysor protein microarrays, are similar to DNAmicroarrays. Protein microarrays are anotherapproach for analyzing large sets of proteins.Protein chips enable researchers to quickly andeasily survey the entire proteome of a cell withinan organism and to automate protein experi-ments. Protein chips look identical, except eachspot corresponds to one of the organism’sthousands of proteins, instead of one of itsgenes. The intensity of the dot indicates theamount of protein present.
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Protein chips were first developed in 2000 byresearchers at Harvard University. Today thereare many companies manufacturing proteinchips using many types of techniques includingspotting and gel methods. The types of proteinchips available include ‘‘lab on a chip’’, anti-body arrays and antigen arrays, as well as a widerange of chips containing ‘‘alternative captureagents’’ such as proteins, substrates, and nucleicacids. Analysis of protein chips comes withmany challenges including dynamic proteinconcentrations, the sheer number of proteins in acell’s proteome, and the understanding of theprobes for each protein. Steps include the read-ing of the protein levels off the chip, and then theuse of computer software to analyze the massiveamounts of data collected (Schena et al. 1995;MacBeath and Schreiber 2000). To examineexpression at protein level and determine quan-titative and qualitative information on proteinsprotein microarray (chips) two components arerequired: spatially addresses molecules thatrecognize individual protein–protein moietiesand second a method to detect the interaction ofthe individual proteins in the mixture with theircorresponding recognition molecules. In thistechnique, purified ligands or recognition mole-cules such as proteins, peptides, antibodies,antigens, aptamers, carbohydrates, or smallmolecules are spotted on a derivatized surfaceand generally used for examining the proteinexpression level for protein profiling and clinicaldiagnostics. Synthetic phage-displayed antibod-ies are used for this purpose but apart fromantibodies any molecule that can specificallyrecognize individual protein with appropriateaffinity and avidity can do so in immobilizedform.
Applications of protein chip experimentsinclude identifying biomarkers for diseases,investigating protein–protein interactions, andtesting for the presence of antibodies in a sam-ple. Protein chips also have applications incancer research, medical diagnostics, homelandsecurity, and proteomics. Many companies,including Biacore, Invitrogen, and Sigma-Aldrich,produce industrial level protein array systems
that can be used for drug discovery and basicbiological research.
Antibody-Pair Protein Arrays
This microarray consists of arrayed capture anti-bodies and appropriate control and orientationelements. The assay is performed by adding anantigen standard or test sample followed by adetector antibody which is modified with adirectly detectable label-enzyme, fluorescentmolecule, isotope, etc., or it is biotinylated fordetection after subsequent probing with labeledstreptavidin. Antibody-pair microarrays are a typeof multiplexed ELISAs. Sandwich-style antibodypair microarrays (Fig. 8.9) can be used for quali-tative or comparative detection of protein analytesor for protein quantification when appropriatestandards are available. Limitation of this tech-nique is that it is difficult to produce specificantibodies for large number of human proteins.
Single Antibody/Labeled Microarray Sample
Microarray
This technique is useful for protein targets, suchas poorly characterized cell signaling proteins,for which paired antibodies are not available.Drawback of this technique is its lack of anti-body redundancy, which helps to ensure specificantigen recognition (Fig. 8.10).
Cellular Lysate Protein Arrays
Microaarays of cellular proteins are eitherarrayed as complex protein mixtures or arrayedas purified or over-expressed proteins. Complexprotein mixture arrays are dot blots of cellularlysates. These microarrays consisting of libraries
Fig. 8.9 Sandwich-style antibody-pair microarray
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of purified or over-expressed proteins allowscreening for protein–protein interactions andkinase activities.
8.5 Post-translationalModifications
The human proteome is more complex than thehuman genome and single genes encode multi-ple proteins. Genomic recombination, tran-scription initiation at alternative promoters,differential transcription termination, and alter-native splicing of the transcript are mechanismsthat generate different mRNA transcripts from asingle gene. The increase in complexity from thegenome to the proteome is further facilitated byprotein post-translational modifications (PTMs).PTMs are chemical modifications that play a keyrole in functional proteomics, because theyregulate activity, localization and interactionwith other cellular molecules such as proteins,nucleic acids, lipids, and cofactors.
PTM increases the functional diversity of theproteome by the covalent addition of functionalgroups or proteins, proteolytic cleavage of reg-ulatory subunits, or degradation of entire pro-teins. These modifications includephosphorylation, glycosylation, ubiquitination,nitrosylation, methylation, acetylation, lipida-tion, and proteolysis and influence almost allaspects of normal cell biology and pathogenesis.PTMs occur at distinct amino acid side chains orpeptide linkages and are most often mediated byenzymatic activity. It is estimated that 5 % ofthe proteome comprises enzymes that perform
more than 200 types of post-translational mod-ifications. These enzymes include kinases,phosphatases, transferases, and ligases, whichadd or remove functional groups, proteins, lip-ids, or sugars to or from amino acid side chains,and proteases, which cleave peptide bonds toremove specific sequences or regulatory sub-units. Many proteins can also modify themselvesusing autocatalytic domains, such as autokinaseand autoprotolytic domains. Post-translationalmodification can occur at any step in the ‘‘lifecycle’’ of a protein. Other modifications occurafter folding and localization are completed toactivate or inactivate catalytic activity or tootherwise influence the biological activity of theprotein. Proteins are also covalently linked totags that target a protein for degradation.Besides single modifications, proteins are oftenmodified through a combination of post-translational cleavage and the addition of func-tional groups through a step-wise mechanism ofprotein maturation or activation.
Protein PTMs can also be reversibledepending on the nature of the modification. Forexample, kinases phosphorylate proteins at spe-cific amino acid side chains, which is a commonmethod of catalytic activation or inactivation.Conversely, phosphatases hydrolyze the phos-phate group to remove it from the protein andreverse the biological activity. Proteolyticcleavage of peptide bonds is a thermodynami-cally favorable reaction and therefore perma-nently removes peptide sequences or regulatorydomains. Consequently, the analysis of proteinsand their post-translational modifications isparticularly important for the study of heartdisease, cancer, neurodegenerative diseases, anddiabetes. The characterization of PTMs providesinsight into the cellular functions underlyingetiological processes.
8.5.1 Types of PTMs
Some of the post-translational modificationshave been described in detail.
Fig. 8.10 Single antibody/labeled microarray samplemicroarray
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8.5.1.1 PhosphorylationReversible protein phosphorylation, principallyon serine, threonine, or tyrosine residues, is oneof the most important and well-studied post-translational modifications. Phosphorylationplays critical roles in the regulation of manycellular processes including cell cycle, growth,apoptosis, and signal transduction pathways.
8.5.1.2 GlycosylationProtein glycosylation has significant effects onprotein folding, conformation, distribution, sta-bility, and activity. Glycosylation encompasses adiverse selection of sugar-moiety additions toproteins that ranges from simple monosaccha-ride modifications of nuclear transcription fac-tors to highly complex branched polysaccharidechanges of cell surface receptors. Carbohydratesin the form of aspargine-linked (N-linked) orserine/threonine-linked (O-linked) oligosaccha-rides are major structural components of manycell surface and secreted proteins.
8.5.1.3 UbiquitinationUbiquitin is an 8-kDa polypeptide consisting of76 amino acids that is appended to the e-aminogroup of a lysine residue on a target protein viathe C-terminal glycine of ubiquitin. Followingan initial monoubiquitination event, the forma-tion of a ubiquitin polymer may occur andpolyubiquitinated proteins are then recognizedby the 26S proteasome that catalyzes the deg-radation of the ubiquitinated protein and therecycling of ubiquitin.
8.5.1.4 S-NitrosylationNitric oxide (NO) is produced by three isoformsof nitric oxide synthase (NOS) and is a chemicalmessenger that reacts with free cysteine residuesto form S-nitrothiols (SNOs). S-nitrosylation is acritical PTM used by cells to stabilize proteins,regulate gene expression, and provide NOdonors, and the generation, localization, activa-
tion, and catabolism of SNOs are tightly regu-lated. S-nitrosylation is a reversible reaction, andSNOs have a short half life in the cytoplasmbecause of the host of reducing enzymes,including glutathione (GSH) and thioredoxin,that denitrosylate protein. Therefore, SNOs areoften stored in membranes, vesicles, the inter-stitial space, and lipophilic protein folds toprotect them from denitrosylation. Only specificcysteine residues are S-nitrosylated.
8.5.1.5 MethylationThe transfer of one-carbon methyl groups tonitrogen or oxygen (N- and O-methylation,respectively) to amino acid side chains increasesthe hydrophobicity of the protein and can neu-tralize a negative amino acid charge when boundto carboxylic acids. Methylation is mediated bymethyltransferases, and S-adenosyl methionine(SAM) is the primary methyl group donor.Methylation occurs so often that SAM has beensuggested to be the most-used substrate inenzymatic reactions after ATP. Additionally,while N-methylation is irreversible, O-methylationis potentially reversible. Methylation is a well-known mechanism of epigenetic regulation, ashistone methylation and demethylation is influ-encing the availability of DNA for transcription.Amino acid residues can be conjugated to asingle methyl group or multiple methyl groupsto increase the effects of modification.
8.5.1.6 N-AcetylationN-acetylation, or the transfer of an acetyl group tonitrogen, occurs in almost all eukaryotic proteinsthrough both irreversible and reversible mecha-nisms. N-terminal acetylation requires the cleav-age of the N-terminal methionine by methionineaminopeptidase (MAP) before replacing theamino acid with an acetyl group from acetyl-CoAby N-acetyltransferase (NAT) enzymes. Thistype of acetylation is co-translational, in thatN-terminus is acetylated on growing polypeptidechains that are still attached to the ribosome.While 80–90 % of eukaryotic proteins are
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acetylated in this manner, the exact biologicalsignificance is still unclear. Lysine acetylationon histone N-termini is a common method ofregulating gene transcription. It is reversible andreduces chromosomal condensation to promotetranscription, and the acetylation of these lysineresidues is regulated by transcription factors thatcontain histone acetyletransferase (HAT) activ-ity. While transcription factors with HATactivity act as transcription co-activators, his-tone deacetylase (HDAC) enzymes are co-repressors that reverse the effects of acetylationby reducing the level of lysine acetylation andincreasing chromosomal condensation. Proteinacetylation can be detected by chromosomeimmunoprecipitation (ChIP) using acetyllysine-specific antibodies or by mass spectrometry,where an increase in histone by 42 mass unitsrepresents a single acetylation.
8.5.1.7 LipidationLipidation is a method to target proteins tomembranes in organelles (endoplasmic reticu-lum [ER], Golgi apparatus, mitochondria), ves-icles (endosomes, lysosomes), and the plasmamembrane. There are four types of lipidation: C-terminal glycosyl phosphatidylinositol (GPI)anchor, N-terminal myristoylation, S-myris-toylation, and S-prenylation. Each type ofmodification gives proteins distinct membraneaffinities, although all types of lipidationincrease the hydrophobicity of a protein and thusits affinity for membranes. The different types oflipidation are also not mutually exclusive, in thattwo or more lipids can be attached to a givenprotein. GPI anchors tether cell surface proteinsto the plasma membrane. This type of modifi-cation is reversible, as the GPI anchor can bereleased from the protein by phosphoinositol-specific phospholipase C. This lipase is used inthe detection of GPI-anchored proteins to releaseGPI-anchored proteins from membranes for gelseparation and analysis by mass spectrometry.N-myristoylation is a method to give proteins ahydrophobic handle for membrane localization.S-palmitoylation adds a C16 palmitoyl groupfrom palmitoyl-CoA to the thiolate side chain of
cysteine residues via palmitoyl acyl transferases(PATs). S-prenylation covalently adds a far-nesyl (C15) or geranylgeranyl (C20) group tospecific cysteine residues within 5 amino acidsfrom the C-terminus via farnesyl transferase(FT) or geranylgeranyl transferases.
8.5.1.8 ProteolysisPeptide bonds are indefinitely stable underphysiological conditions, and therefore cellsrequire some mechanism to break these bonds.Proteases comprise a family of enzymes thatcleave the peptide bonds of proteins and arecritical in antigen processing, apoptosis, surfaceprotein shedding, and cell signaling. The familyof over 11,000 proteases varies in substratespecificity, mechanism of peptide cleavage,location in the cell, and the length of activity.Proteases can generally be separated into groupsbased on the type of proteolysis. Degradativeproteolysis is critical to remove unassembledprotein subunits and misfolded proteins and tomaintain protein concentrations at homeostaticconcentrations by reducing a given protein to thelevel of small peptides and single amino acids.Proteases also play a biosynthetic role in cellbiology that includes cleaving signal peptidesfrom nascent proteins and activating zymogens,which are inactive enzyme precursors thatrequire cleavage at specific sites for enzymefunction.
Proteolysis is a thermodynamically favorableand irreversible reaction. Therefore, proteaseactivity is tightly regulated to avoid uncontrolledproteolysis through temporal and/or spatialcontrol mechanisms including regulation bycleavage in cis or trans and compartmentaliza-tion (e.g., proteasomes, lysosomes). The diversefamily of proteases can be classified by the siteof action, such as aminopeptidases and car-boxypeptidase, which cleave at the amino orcarboxy terminus of a protein, respectively.Another type of classification is based on theactive site groups of a given protease that areinvolved in proteolysis, e.g., Serine proteases,Cysteine proteases, Aspartic acid proteases, andZinc metalloproteases.
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8.5.2 Methods for Study of PTMs
Methods such as phosphoproteomics and gly-coproteomics are used to study post-translationalmodifications.
8.5.2.1 GlycoproteomicsIt is now widely accepted that correct proteinglycosylation is important for the normal func-tion of many cellular and secreted proteins.Glycosylation has been shown to be necessaryfor many diverse aspects of protein function, forexample, to ensure correct protein folding, toprotect proteins from proteolytic attack, as rec-ognition molecules for protein clearance fromthe serum, and as adhesion molecules. In parallelto the role of glycosylation in normal proteinfunction, the number of examples in whichchanges occur to the glycosylation of proteins indisease expands.
The standard approach for the identificationof (glyco) proteins after separation of the pro-teome by 1D- or 2D-PAGE is generally byMALDI-MS fingerprint analysis. The separatedproteins are digested, in-gel, with a proteasesuch as trypsin, analyzed on a mass spectrometerand the resulting masses are compared withthose generated by the theoretical digestion ofprotein sequences in databases (SWISSPROTand NCBI). Using this approach, a proportion ofpeptide masses may not be identified as origi-nating from the proteolytic digestion of theprotein. These difficulties in protein identifica-tion are often explained by the occurrence ofpost-translational modifications such as glyco-sylation and phosphorylation, which are notpredicted by the majority of databases whosesequences, in the main, have been determinedfrom the characterization of cDNAs. Computa-tional tools, such as FindMod, Peptldent, andPeptide-Mass (http://www.expasy.ch/tools/) cancalculate the difference between the observedand predicted masses giving a useful insight intothe types of possible modifications that mayhave occurred to the protein. Another approachto identify likely O-link glycosylation sites
utilizes a neural network algorithm and incor-porates into an O-GlycBase database (http://www.cbs.dtu.dk/databases/OGLYCBASE/Oglyc.base.html), which provides free access and informa-tion regarding the type of glycan, the glycosyl-ation site, species from which the glycoproteinwas derived, etc. To determine whether or not aprotein is glycosylated is to use the periodicacid-Schiff (PAS) stain. Combining the periodicacid reaction with fluorescent dye technologyenables the detection of glycoproteins at nano-gram sensitivity. Proteins separated by 2D-PAGE can be transferred to inert mem-branes, usually made of either nitrocellulose orpolyvinylidene difluoride, by a process known asWestern blotting. The glycosylation of the pro-teins can then be examined by detecting themonosaccharides that are present by using car-bohydrate-binding proteins, for example, lectins,or antibodies. The sensitivity of detection ofglycan residues may be enhanced to nanomolconcentrations by the use of biotin conjugatedlectins and/or antibodies using streptavidin-horse radish peroxidase, to produce either acolored substrate or a light reaction. Most of themethods for detailed analysis of N-linked gly-cans involve the removal of the glycan moietyfrom the protein backbone first, although therehave been recent reports of analysis of intactglycoproteins after separation by 2D-PAGE.
The most frequently used approach for glycanrelease is endoglycosidase digestion and for thisPNGase-F (peptide N-glycosidase F) is used.Enzymatic treatment is favored over chemicalmethods, such as hydrazinolysis or j-elimination,for the release of N-linked sugars because theglycans are less extensively modified and theprotein can also be recovered intact. Chemicalmethods such as hydrazinolysis and, more usually,non-reductive j-elimination or trifluoromethane-sulfonic acid are employed. Glycan analysis isusually attempted for glycoproteins that areavailable in microgram quantities. Once released,various analytical techniques are available for thecharacterization of carbohydrate structures, e.g.,chromatography, electrophoresis, and massspectrometry. Web-based Bioinformatic analysis
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tools are available that enable the interpretation ofsugar mass spectrometric data, e.g., GlycoModand GlycoSuiteDB (www.expasy.ch/tools/glycomod and http://tmat.proteomesystems.com/glycosuite/).
8.5.2.2 PhosphoproteomicsPhosphoproteomics deals with the identificationand quantification of phosphorylated proteinsand identification of phosphorylation sites. Pro-tein kinases control every basic cellular processincluding metabolism, growth, division, differ-entiation, motility, immunity learning, andmemory via regulated protein phosphorylation.Mass spectrometry is the best method used forthe identification of phosphorylation sites. Onemost common strategy for enrichment of phos-phoproteomes is a combination of chromatog-raphy with affinity-based enrichment. Thesechromatographic techniques include immobi-lized metal affinity chromatography (IMAC),immunoprecipitation, metal oxide affinity chro-matography (MOAC). In addition antibodiesagainst phosphoaminoacid epitopes, magneticmaterials, and nanoparticles as well as metal ionphosphopeptide precipitations are used foraffinity-based enrichment of phosphopeptides.IMAC uses metal chelators to immobilize metalions.
Identification of phosphopeptides and phos-phorylation sites is based on database searches.Quantification of site-specific phosphorylation ismeasured in an MS assay. Occupancy of phos-phorylation sites describes the level of phos-phorylation compared with the unmodified sites.
8.6 Methods to Find Proteinsthat Function Together
Some proteins act alone, and the function ofmany of these has been known for years. Butprobably the majority of the proteins in a cell actin concert with others, e.g., the 79 proteins in theeukaryotic ribosome, the *145 proteins in the
spliceosomes, and the myriad transcription fac-tors in the cell.
8.6.1 Affinity Chromatography
For affinity chromatography, the proteins whosepartners are to be found are attached to a solidmatrix in a glass column. A solution containinga mix of possible partners is run through thecolumn. Those proteins that can bind to thetarget will stick; the others will flow through. Abuffer is passed through the column whichweakens the binding interactions. The partnersare washed out and can be identified.
8.6.2 The Yeast Two-Hybrid System
The yeast two-hybrid system is an in vivo sys-tem that utilizes the binding domain (BD) andactivation domain (AD) of genes that are used intranscription, to determine protein interactions.Proteins are fused to both the BD and the AD ofa reporter gene, one to each. When the proteinattached to BD interacts with the protein fused tothe AD, transcription is initiated, and thereporter gene is transcribed. The effect is quan-tifiable by measuring the levels of reporter geneexpressed. The budding yeast, Saccharomycescerevisiae provides an excellent tool for dis-covering protein partners as it can easily betransformed with plasmids containing foreignDNA sequences, can live in either the haploid ordiploid condition and haploid cells can fuse toform diploid cells if they are of opposite matingtypes. Using the two-hybrid method, it has beenpossible to identify many sets of interactingproteins in yeast and other organisms.
For this, using recombinant DNA methods, aplasmid is created containing (1) the DNAencoding the DNA-binding domain of a tran-scription factor needs to turn on expression of areporter gene such as the lacZ gene that encodesthe enzyme b-galactosidase coupled to (2) theDNA encoding the ‘‘target’’ protein, i.e., the
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protein whose possible partners are to beidentified.
The plasmid is inserted into living haploidyeast of one mating type.1. Using the same methods, many different
plasmids are created each containing (a) theDNA encoding the activation domain of thetranscription factor, (b) the DNA encoding apossible partner (‘‘bait’’) protein, and (c) eachof these plasmids is inserted into a yeast cellsand grown as separate clones.
2. Each a clone is mated with the target clone.3. If the fusion protein produced by the tran-
scription and translation of a ‘‘bait’’-con-taining plasmid can bind to the fusion proteincontaining the target, the two domains of thetranscription factor can interact to turn onexpression of the reporter gene (lacZ).
4. They are then grown on an indicator substrateas a result the colonies turn blue.
5. The DNA in these colonies can then be iso-lated and sequenced.
6. Finally the identification of the proteins thatcan associate with the target protein can bedone.
8.6.3 Phage Display
This method involves a DNA bacteriophage thatinfects E. coli and its ability to remain infectiouseven if one of its coat proteins contains segmentsof a foreign protein. The method involvestransformation of bacteriophages with a randommix of DNA from the organism of interestcoupled to the DNA encoding one of the viralcoat proteins. Then the E. coli is infected withthese phages. As the viruses replicate, they notonly propagate the recombinant gene but alsoexpress it as a coat protein. Both will be incor-porated into new virions. The mix of viruses isinfected. The mixture is passed through anaffinity chromatography column to which the‘‘target’’ protein has been fixed. Those virusesthat display a piece of foreign protein (peptide)that can bind to the target will stick to it. Thebound phage is eluted with a buffer. E. coli is
infected and separate colonies (clones) aregrown. The coat protein gene is sequenced tofind the sequence of the foreign DNA inserted init. Using the genetic code dictionary, the aminoacid sequence of the peptide is determined. Thedatabases are searched for a protein containingthis sequence. As a result a protein that associ-ates with the target protein is obtained.
8.6.4 Three-Dimensional Structure
The clearest picture of how different proteinsinteract with one another to form functionalcomplexes will come from determining the 3Dstructure of the complex. There are two methodsused for this, X-ray crystallography and NMRspectroscopy. X-ray crystallography requirescrystallization of the protein. This is often dif-ficult especially for complexes of two or moreproteins. NMR spectroscopy has been especiallyuseful in producing 3D images of proteins thatcannot be crystallized.
8.7 Proteoinformatics/Bioinformatics in Proteomics
Proteoinformatics is the use of bioinformaticsand computational biology techniques solelywithin the realm of protein identification andproteomics. Proteoinformatics is currently in itsinfancy and the largest work being done is onstandardizing databases and data submission.Other proteoinformatic work is being done onthe image analysis of 2D gels and other imagesin proteomics used to help identify and annotateproteins in the proteome. An increased ability togenerate large amounts of data from the use ofhigh-throughput methods has led to an increasedreliance on computers for data acquisition,storage, and analysis. The internet has alsoenabled collaboration and sharing of data thatwould not have been possible, leading to thedevelopment of large public databases withcontributors all over the world. Many databasesexist for protein-related information, e.g.,
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protein data bank (PDB) which handles structureand sequence information for proteins with adetermined crystal structure. Expasy is a popularand well-curated resource for proteomics dat-abases and tools, including resources such as theProsite protein feature and domain database,protein BLAST (Basic Local Alignment andSearch Tool, for similarity searching) andstructure prediction. NCBI also provides manyresources for proteins (Binz et al. 2004). As withother bioinformatics resources, ‘‘in silico’’ dis-covery is not meant as a replacement for labtechniques, but a supplement to work done in awet lab.
8.7.1 Protein Identification Database
Protein Identifications Databases such as Pro-Found at Rockefeller University and ProteinProspector at the UCSF Mass SpectrometryFacility are used to identify proteins found withproteomics techniques such as mass spectrome-try. Digestion of proteins into peptide fragmentsallows each protein to break apart in a differentway, resulting in a unique peptide fingerprintthat can be used to identify the protein. Themasses of these fragments as well as themolecular weights and isoelectric points arestored in many of these databases. This data canbe used to perform high-throughput proteinidentification. Protein databases are more spe-cialized than primary sequence databases. Theycontain information derived from the primarysequence databases. Some contain proteintranslations of the nucleic acid sequences. Somecontain sets of patterns and motifs derived fromsequence homologs (Tables 8.2 and 8.3).
8.8 Applications of Proteomics
The goal of proteomics is to analyze the varyingproteomes of an organism at different times, inorder to highlight differences between them.Proteomics analyzes the structure and functionof biological systems. For example, the protein
content of a cancerous cell is often differentfrom that of a healthy cell. Certain proteins inthe cancerous cell may not be present in thehealthy cell, making these unique proteins goodtargets for anti-cancer drugs. Both purificationand identification of proteins in any organism isaffected by a multitude of biological and envi-ronmental factors.
8.8.1 Drug Discovery
The process of drug discovery is quite complex,integrating many disciplines, including structuralbiology, metabolomics, proteomics, and com-puter science. The process includes target selec-tion, lead identification, and preclinical andclinical candidate selection (Okerberg et al. 2005).Proteomics can be used for identifying proteinsinvolved in disease pathogenesis and physiologi-cal pathway reconstruction facilitates the everincreasing discovery of new, novel drug targets,their respective modes of action mechanistically,and their biological toxicology. While the causesof many clinical problems vary greatly in theirnature and origin, in some cases, the cause is foundat the protein level, involving protein function,protein regulation, or protein–protein interac-tions. For example alkaptonuria is characterizedby a defect in the gene coding for the enzymehomogentisic acid oxidase, inhibiting the metab-olism of homogentisic acid to maleylacetoaceticacid, within the phenylalanine degradation path-way. The cause of this inborn disease is due to asingle gene genetic defect, the clinical manifes-tations, which include excretion of black urine, area function of the built up of homogentisic acidresulting from a defective enzyme (protein).
Virtual drug libraries are being developed,both in the public and private sectors. Thesedatabases contain potential drug compounds;these compounds may or may not exist outsideof a computer database and new compoundsdeveloped through various methods of synthesisare continually added. Methods of modifyingexisting database entries to create new isomersand derivatives are also used, to more
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Table 8.2 Some protein databases available on the Internet
S.N. Database Description
1. PIR Protein information resource—a comprehensive, non-redundant, expertlyannotated, fully classified, and extensively cross-referenced protein sequencedatabase
2. SWISS-PROT andTrEMBL
SWISS-PROT is a curated protein sequence database. TrEMBL is a computer-annotated supplement of SWISS-PROT that contains all the translations ofEMBL nucleotide sequence entries not yet integrated in SWISS-PROT
3. TIGR A collection of curated databases containing DNA and protein sequence, geneexpression, cellular role, protein family, and taxonomic data for microbes, plantsand humans
4. ALIGN A compendium of sequence alignments: it is a companion resource to PRINTS
5. BLOCKS Multiply aligned ungapped segments corresponding to the most highly conservedregions of proteins
6. DOMO A database of homologous protein domain families
7. HOMSTRAD A curated database of structure-based alignments for homologous proteinfamilies
8. Pfam A database of multiple alignments of protein domains or conserved proteinregions. The alignments represent some evolutionary conserved structure whichhas implications for the protein’s function. Profile hidden Markov models (profileHMMs) built from the Pfam alignments can be very useful for automaticallyrecognizing that a new protein belongs to an existing protein family, even if thehomology is weak
9. ProSite A database of protein families and domains consisting of biologically significantsites, patterns, and profiles
10. Library of ProteinFamily Cores
Structural alignments of protein families and computed average core structuresfor each family. Useful for building models, threading, and exploratory analysis
11. ModBase A database of 3D protein models calculated by comparative modeling
12. RCSB proteindatabank
Single international repository for the processing and distribution of 3Dmacromolecular structure data primarily determined experimentally
13. Protein loopclassification
Conformational clusters and consensus sequences for protein loops derived bycomputational analysis of their structures
14. CluSTr Offers an automatic classification of UniProtKB/Swiss-Prot ? UniProtKB/TrEMBL
15. CSA The Catalytic Site Atlas (CSA) is a resource of catalytic sites and residuesidentified in enzymes using structural data
16. InterPro The InterPro database is an integrated documentation resource for proteinfamilies, domains, and functional sites
17. IPI International Protein Index contains a number of non-redundant proteome sets ofhigher eukaryotic organisms constructed from UniProtKB/Swiss-Prot,UniProtKB/TrEMBL, Ensemb,l and RefSeq
18. PANDIT PANDIT—protein and associated nucleotide domains with Inferred Trees.PANDIT is a collection of multiple sequence alignments and phylogenetic treescovering many common protein domains.
19. Patent Data Resources Patent data resources at the EBI contain patent abstracts, patent chemicalcompounds, patent sequences, and patent equivalents. There are various ways ofaccessing and searching the patent data
20. UniProt The Universal Protein Resource for protein sequences and is the central hub forthe collection of functional information on proteins, with accurate, consistent,and rich annotation, the amino acid sequence, protein name or description,taxonomic data, and citation information
21. UniProt Archive A non-redundant archive of protein sequences extracted from public databasesand contains only protein sequences
(continued)
8 Proteomics 143
adequately cover a range of potential drugcompounds. Docking and scoring are imple-mented using known and hypothetical drug tar-gets on a protein, coupled with the databases ofvirtual chemical compounds. After docking,scoring is carried out using mathematical mod-els. These models determine the chemicalbinding strength and energy state of the drug-protein complex. Those hits with high rankingscores are subsequently subjected to in vivotests; hits with positive scores in both areas arethen known to be leads. Evaluation of dockedand scored complexes are then made, selectingan arbitrary number of top hits to be furtherscreened manually. The first two steps are doneentirely in silico; however, the best complexesnow need to be examined using software visu-alization, often in 3D setups. The compoundsthat make it through docking, scoring, andevaluation become drug leads, and are thenpassed on to undergo drug testing techniques ina wet lab, to ensure that only compounds witheffects relatively unique to the target system andsafe to the rest of organism are considered.
8.8.2 Protein Biomarkers
Biomarkers of drug efficacy and toxicity arebecoming a key need in the drug developmentprocess. Mass spectral-based proteomic tech-nologies are ideally suited for the discovery ofprotein biomarkers in the absence of any priorknowledge of quantitative changes in proteinlevels. The success of any biomarker discoveryeffort will depend upon the quality of samplesanalyzed, the ability to generate quantitativeinformation on relative protein levels, and theability to readily interpret the data generated.Some diseases which have protein biomarkersthat show promise as a screening tool are breastcancer, Alzheimer’s, leukemia, Amyotrophiclateral sclerosis, and Parkinson’s. A series of sixsteps must be accomplished in order to suc-cessfully validate a biomarker or set of bio-makers: discovery, qualification, verification,assay optimization, validation, and commer-cialization. Once a biomarker is found andaccepted, it can be used to possibly predict andprevent the disease it is related to.
Table 8.2 (continued)
S.N. Database Description
22. UniProt/UniRef Features clustering of similar sequences to yield a representative subset ofsequences. This produces very fast search times
23. UniProtKB-GOA Provides assignments of proteins in UniProtKB/Swiss-Prot, UniProtKB/TrEMBL, and IPI to the Gene Ontology resource
24. UniProtKB/Swiss-Prot
An annotated protein sequence database. Part of the UniProtKB
25. UniProtKB/TrEMBL A computer generated protein database enriched with automated classificationand annotation. Part of the UniProtKB
26. UniProt/UniMES A repository specifically developed for metagenomic and environmental data
27. UniSave The UniProtKB sequence/annotation version archive (UniSave) is a repository ofUniProtKB/Swiss-Prot and UniProtKB/TrEMBL entry versions
28. Phospho.ELM Database of experimentally verified phosphorylation sites in eukaryotic proteins
29. DMPC Database of membrane protein Contacts
30. BioGRID Database of protein and genetic interactions
31. PROSITE Database of protein families and domains
32. IPI International protein index
33. HPRD Human protein reference database
34. CDD NCBI conserved domain database
35. ConSurf-DB Evolutionary conservation profiles of protein structures
144 I. Ravi
Table 8.3 Some protein databases available on the Internet used for performing specific functions
S.N. Database Description
Identification and characterization with peptide mass fingerprinting data
1. FindMod Predict potential protein post-translational modifications and potentialsingle amino acid substitutions in peptides. Experimentally measuredpeptide masses are compared with the theoretical peptides calculatedfrom a specified Swiss-Prot entry or from a user-entered sequence, andmass differences are used to better characterize the protein of interest
2. FindPept Identify peptides that result from unspecific cleavage of proteins fromtheir experimental masses, taking into account artifactual chemicalmodifications, post-translational modifications (PTM), and proteaseautolytic cleavage
3. Mascot Peptide mass fingerprint from Matrix Science Ltd., London
4. PepMAPPER Peptide mass fingerprinting tool from UMIST, UK
5. ProFound Search known protein sequences with peptide mass information fromRockefeller and NY Universities [or from Genomic Solutions]
Identification and characterization with MS/MS data
6 QuickMod Open modification spectral library search tool for identification ofMS/MS data
7. Phenyx Protein and peptide identification/characterization from MS/MS datafrom GeneBio, Switzerland
8. Mascot Sequence query and MS/MS ion search from Matrix Science Ltd.,London
9. OMSSA MS/MS peptide spectra identification by searching libraries of knownprotein sequences
10. PepFrag Search known protein sequences with peptide fragment massinformation from Rockefeller and NY Universities
11. ProteinProspector UCSF tools for fragment-ion masses data (MS-Tag, MS-Seq,MS-Product, etc.)
Identification with isoelectric point, molecular weight, and/or amino acid composition
12. AACompIdent Identify a protein by its amino acid composition
13. AACompSim Compare the amino acid composition of a UniProtKB/Swiss-Prot entrywith all other entries
14. TagIdent Identify proteins with isoelectric point (pI), molecular weight (Mw)and sequence tag, or generate a list of proteins close to agiven pI and Mw
15. MultiIdent Identify proteins with isoelectric point (pI), molecular weight (Mw),amino acid composition, sequence tag, and peptide mass fingerprintingdata
Other prediction or characterization tools
16. ProtParam Physico-chemical parameters of a protein sequence (amino acid andatomic compositions, isoelectric point, extinction coefficient, etc.)
17. Compute pI/Mw Compute the theoretical isoelectric point (pI) and molecular weight(Mw) from a UniProt Knowledgebase entry or for a user sequence
18. PeptideCutter Predicts potential protease and cleavage sites and sites cleaved bychemicals in a given protein sequence
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8 Proteomics 145
8.8.3 Study of Tumor Metastasis
Tumor metastasis is the dominant cause of deathin cancer patients. However, the molecular andcellular mechanisms underlying tumor metasta-sis are still elusive. The identification of proteinmolecules with their expressions correlated to
the metastatic process help to understand themetastatic mechanisms and thus facilitate thedevelopment of strategies for the therapeuticinterventions and clinical management of can-cer. Proteomic technology has been widely usedin biomarker discovery and pathogenetic studiesincluding tumor metastasis.
Table 8.3 (continued)
S.N. Database Description
19. PeptideMass Calculate masses of peptides and their post-translational modificationsfor a UniProtKB/Swiss-Prot or UniProtKB/TrEMBL entry or for a usersequence
20. xComb Computes all possible crosslinks based on proteins of interest forfurther interrogation using standard search engine
21. xQuest Search machine to identify cross-linked peptides from complexsamples and large protein sequence databases
22. SmileMS Small molecule identification
Other proteomics tools-glycotools
23. GlycanMass Calculate the mass of an oligosaccharide structure
24. GlycoMod Predict possible oligosaccharide structures that occur on proteins fromtheir experimentally determined masses (can be used for free orderivatized oligosaccharides and for glycopeptides)
25. GlycospectrumScan an analytical tool independent of MS-platform that accuratelyidentifies and assigns the oligosaccharide heterogeneity onglycopeptides from MS data of a mixture of peptides andglycopeptides
26. Glycoviewer a visualization tool for representing a set of glycan structures as asummary figure of all structural features using icons and colorsrecommended by the Consortium for Functional Glycomics (CFG)
Other tools for MS data (vizualization, quantitation, analysis, etc.)
27. HCD/CID spectra merger a tool to merge the peptide sequence-ion m/z range from CID spectraand the reporter-ion m/z range from HCD spectra into the appropriatesingle file, to be further used in identification and quantification searchengines
28. MALDIPepQuant Quantify MALDI peptides (SILAC) from Phenyx output
29. MSight Mass spectrometry imager
Other tools for 2-DE data (image analysis, data publishing, etc.)
30. ImageMaster/Melanie Software for 2-D PAGE analysis
31. Make2D-DB II A package to build a web-based proteomics database
DNA ? Protein
32. Translate Translates a nucleotide sequence to a protein sequence
33. Transeq Nucleotide to protein translation from the EMBOSS package
34. (Reverse)-Transcription andtranslation tool genewise
Compares a protein sequence to a genomic DNA sequence, allowingfor introns and frameshifting errors
35. Reverse translate Translates a protein sequence back to a nucleotide sequence
36. BCM search launcher Six frame translation of nucleotide sequence(s)
146 I. Ravi
8.8.4 Neurotrauma
Proteomics is a burgeoning field that may pro-vide a valuable approach to evaluate the post-traumatic central nervous system (CNS) and thenewer proteomic technologies that are availableto most researchers in neurotrauma. Proteomicswill likely be very useful for developing diag-nostic predictors after CNS injury and for map-ping changes in proteins after injury in order toidentify new therapeutic targets. Neurotraumaresults in complex alterations to the biologicalsystems within the nervous system, and thesechanges evolve over time. Exploration of the‘‘new nervous system’’ that follows injury willrequire methods that can both fully assess andsimplify this complexity.
8.8.5 Renal Disease Diagnosis
In the diagnosis and treatment of kidney disease,a major priority is the identification of disease-associated biomarkers. Proteomics, with itshigh-throughput and unbiased approach to theanalysis of variations in protein expression pat-terns (actual phenotypic expression of geneticvariation), is most suitable for biomarker dis-covery. Combining such analytical techniques as2D gel electrophoresis with MS, has enabledconsiderable progress to be made in cataloguingand quantifying proteins present in urine andvarious kidney tissue compartments in bothnormal and diseased physiological states.
8.8.6 Neurology
In neurology and neuroscience, many applica-tions of proteomics have involved neurotoxi-cology and neurometabolism, as well as in thedetermination of specific proteomic aspects ofindividual brain areas and body fluids in neuro-degeneration. Investigation of brain proteingroups in neurodegeneration, such as enzymes,cytoskeleton proteins, chaperones, synaptosomalproteins, and antioxidant proteins, is in progress
as phenotype-related proteomics. The concomi-tant detection of several hundred proteins on agel provides sufficiently comprehensive data todetermine a pathophysiological protein networkand its peripheral representatives. An additionaladvantage of advanced proteomics technology isthat unknown proteins have been identified asbrain proteins.
8.8.7 Fetal and Maternal Medicine
The expression levels of many proteins stronglydepend on complex, but well-balanced regula-tory systems. The proteome, unlike the genome,is highly dynamic. This variation depends on thebiological function of a cell, but also on signalsfrom its environment. In (bio) medical researchit has become increasingly apparent that cellularprocesses, in particular in disease, are deter-mined by multiple proteins. Hence, it is impor-tant not to focus on one single gene product (oneprotein), but to study the complete set of geneproducts (the proteome). In this way the multi-factorial relations underlying certain diseasesmay be unraveled potentially identifying thera-peutical targets. For many diseases, character-ization of the functional proteome is crucial forelucidating alterations in protein expression andmodifications. Abnormalities in splicing or post-translational modifications can cause a diseaseprocess but they can also be a consequence. Anexample is that patients with diabetes have highblood glucose which glycosylates hundreds oreven thousands of proteins, including HbA1cwhich is used to monitor diabetic control.
8.8.8 Urological Cancer Research
Proteomic analysis allows the comparison of theproteins present in a diseased tissue sample withthe proteins present in a normal tissue sample.Any proteins, which have been altered eitherquantitatively or qualitatively between the nor-mal and diseased sample are likely to be asso-ciated with the disease process. These proteins
8 Proteomics 147
can be identified and may be useful as diagnosticmarkers for the early detection of the disease orprognostic markers to predict the outcome of thedisease or they may be used as drug targets forthe development of new therapeutic agents.There are potential future applications of pro-teomic analysis in urological cancer research.
8.8.9 Autoantibody Profilingfor Study and Treatmentof Autoimmune Disease
Proteomics technologies enable profiling ofautoantibody responses using biological fluidsderived from patients with autoimmune disease.They provide a powerful tool to characterizeautoreactive B-cell responses in diseasesincluding rheumatoid arthritis, multiple sclerosis,autoimmune diabetes, and systemic lupus ery-thematosus. Autoantibody profiling may servepurposes including classification of individualpatients and subsets of patients based on their‘autoantibody fingerprint’, examination of epi-tope spreading and antibody isotype usage, dis-covery and characterization of candidateautoantigens, and tailoring antigen-specifictherapy. In future, proteomics technologies willbroaden understanding of the underlying mech-anisms of and will further our ability to diagnose,prognosticate, and treat autoimmune disease.
8.8.10 Cardiovascular Research
The development of proteomics is a timely onefor cardiovascular research. Analyses at theorgan, subcellular, and molecular levels haverevealed dynamic, complex, and subtle intra-cellular processes associated with heart andvascular diseases. Establishment of species- andtissue-specific protein databases provides afoundation for subsequent proteomic studies.Evolution of proteomic techniques has permittedmore thorough investigation into molecularmechanisms underlying cardiovascular disease,facilitating identification not only of modified
proteins but also of the nature of their modifi-cation. Continued development should lead tofunctional proteomic studies, in which identifi-cation of protein modification, in conjunctionwith functional data from established biochem-ical and physiological methods, has the ability tofurther our understanding of the interplaybetween proteome change and cardiovasculardisease.
8.8.11 Diabetes Research
Proteomics is the investigation of all the proteinsand their various modifications making up asystem, a cell, tissue, or organism. The tech-niques involved in proteomics allow the globalscreening of complex samples of proteins andprovide qualitative and quantitative evidence ofaltered protein expression. This lends itself tothe investigation of the molecular mechanismsunderlying disease processes and the effects oftreatment. This can thus be applied to diabetesresearch.
8.8.12 Nutrition Research
Proteomics holds great promise for discoveriesin nutrition research, including profiles andcharacteristics of dietary and body proteins;digestion, absorption, and metabolism of nutri-ents; functions of nutrients and other dietaryfactors in growth, reproduction and health; bio-markers of the nutritional status and diseases;and individualized requirements of nutrients.The proteome analysis is expected to play animportant role in solving major nutrition-asso-ciated problems in humans and animals, such asobesity, diabetes, cardiovascular disease, cancer,aging, and intrauterine fetal retardation.
8.8.13 Plant Physiology
Although significant advances in the compre-hensive profiling, functional analysis, and
148 I. Ravi
regulation of proteins has occurred in modelorganisms such as yeast (Saccharomyces cere-visiae) and in humans, proteomics research inplants has not advanced at the same pace. Theavailability of the complete Arabidopsis (Ara-bidopsis thaliana) genome, which is smallcompared to that of other plants, along with anincreasingly comprehensive catalog of protein-coding information from large-scale cDNAsequencing and transcript mapping experiments,set it as a complex model organism to studyplant proteomics. The application of proteomicapproaches to plants involves (1) comprehensiveidentification of proteins, their isoforms, andtheir prevalence in each tissue; (2) characteriz-ing the biochemical and cellular functions ofeach protein; (3) the analysis of protein regula-tion and its relation to other regulatory networks.
8.9 Conclusion
The post-genomic era requires the completeunderstanding of the protein structure andfunction. Proteomics is the study of the set ofproteins produced (expressed) by an organism,tissue, or cell and the changes in the proteinexpression patterns in different environmentsand conditions. It has been classified intostructural, expression, and functional proteo-mics. A number of analytical techniques used inproteomics study are 2D gel electrophoresis,chromatography, MS, NMR, X-ray crystallog-raphy, ICAT, FRET, Yeast two-hybrid system,etc. The sequencing of the human genome hasincreased interest in proteomics because DNAsequence information provides only a view ofthe various ways in which the cell might use itsproteins, whereas proteomics includes identifi-cation of proteins in biological tissues, charac-terization of their physicochemical properties,and description of their behavior. This new data
set holds great promise for proteomic applica-tions in science, medicine, and mainlypharmaceuticals.
References
Andersen JS, Svensson B, Roepstorff P (1996) Electro-spray ionization and matrix assisted laser desorptionionization mass spectrometry: powerful analyticaltools in recombinant protein chemistry. Nat Biotech-nol 14:449–457
Binz PA, Gasteiger E, Sanchez J, Bairoch A, Hochst-rasser DF, Appel, RD (2004) Proteome analysis. In:Lengauer Th (ed) Bioinformatics. from Genomes toDrugs. Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim, FRG. ISBN:3-527-29988-2, pp 69–118
Cagney G, Amiri S, Premawaradena T, Lindo M, Emili A(2003). In silico proteome analysis to facilitateproteomics experiments using mass spectrometry.Proteome Science 1:5. http://www.proteomesci.com/content/1/1/5 (Open Access article)
Garret RH, Grisham CM (1995) Proteins: their biologicalfunctions and primary structure. In: BiochemistryGareet RH, Grisham CM (eds) Saunders CollegePublishing, Harcourt Brace College Publishers, Phil-adelphia, pp 81–131
Graves PR, Haystead AJ (2002) Molecular biologistsguide to proteomics. Microbiol Mol Biol Rev66(1):39–63
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Okerberg ES, Wu J, Zhang B, Sammi B, Blackford K,Winn DT, Shreder KR, Burbaum JJ, Palricelli MP(2005) High resolution functional proteomics byactive site peptide profiling. PNAS 102(14):4996–5001
Schena M, Shalon D, Davis RW, Brown PO (1995)Quantitative monitoring of gene expression patternswith a complementary DNA microarray. Science270(5235):467–470
Yates JR, Ruse CI, Nakorchevesky A (2009) Proteomicsby mass spectrometry: approaches, advances andapplications. Annu Rev Biomed Eng 11:49–79
8 Proteomics 149
9Recent Advances in Stem Cell Research
Shweta Kulshreshtha and Pradeep Bhatnagar
Abstract
Stem cells are totipotent progenitor cells which are capable of self-renewal and differentiation into multilineage cell types. The stem cellsdivide unlimitedly; therefore these are ideal targets for in vitromanipulation. Researchers have been studying the biology of stem cellsto find new ways of treating various diseases. They provide nearlylimitless potential for medical applications due to having ability ofproducing multilineage cell types. Although early researches havefocused on hematopoietic stem cells (HSCs), these have also been foundto be present in various other tissues. This chapter focuses on the stemcell technology and the potential of stem cells in treating variousdiseases. Also, the current researches and the clinical status of treatmentsbased on stem cells are discussed.
9.1 Introduction
In the new millennium, biological and biotech-nological approaches have been extensivelyexplored to find out ‘‘biological solutions tobiological problems.’’ With the extraordinaryadvances taking place in the field of cellular andmolecular biology, we are on the verge of
finding simple mechanical care to consider bio-logical solutions to health promotion, riskassessment, diagnosis, treatment, and prognosis.It is only in the last decade that an informationexplosion occurred in the area of stem cellresearch. Stem cells are likely to revolutionizethe entire health care delivery. This is a hightime to familiarize ourselves with stem cells,their characteristics, potential applications, cur-rent research and therapy, and possible barriersof their applications.
Stem cells, found mostly in multicellularorganisms, can be defined as primitive, generic,and undifferentiated form of cells which arelocated in fetus, embryo, and adult body and arecharacterized by their ability to renew them-selves through mitotic cell division and differ-entiate into a diverse range of specialized celltypes. They can also build special tissues in the
S. Kulshreshtha (&)Amity Institute of Biotechnology, Amity UniversityRajasthan, Jaipur, Indiae-mail: [email protected]
P. BhatnagarDepartment of Life Sciences, The IIS University,Mansarovar, Jaipur, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_9, � Springer India 2014
151
body and can be grown in vitro for use inresearch or medicine.
Stem cells are important for living organismsfor many reasons. Different types of stem cellssuch as embryonic, induced pluripotent, fetal,and adult are useful in understanding humandevelopment and diseases. Human developmentcan be understood by determining how humanembryonic stem (ES) cells give rise to 220 typesof cells in the body. Before using human EScells, mouse was used as research model fordevelopmental studies. However, developmentalstudies conducted in mice cannot be useful inunderstanding human development due to thedifferences in events of early development.Using ES cells, scientists can witness the veryfirst events in human development to understandhow normal development unfolds and how theprocess may go awry and lead to pregnancy loss,birth defects, and other problems.
Stem cells are also helpful in developingnovel therapies and screening new therapeuticcompounds. They offer enormous promise to getrid of diseases that rob the joy of life and evenlife. Besides, these cells provide a powerfulsource of cells for basic experimental studiesthat can be done in a laboratory, e.g., studyingand characterizing the particular genes andspecialized cell types.
9.2 Historical Developments
In 1981, a British research team succeeded forthe first time in isolating and culturing cells fromearly mouse embryos and also to keep them inundifferentiated form. Embryonic cells, isolatedfrom a brown mouse, were introduced into 4 to7-days-old embryo of a white mouse and it wasfound that offsprings were born with a whitecoat with brown tufts, which were called aschimeras. This proved that cells of all organsoriginated from the injected embryonic cellswhich have totipotency. These cells can also beinduced to differentiate into many different celltypes in in vitro tissue culture and providepotentially unlimited source of raw material fordamaged tissue. They act as an ideal model
system to investigate the factors influencing thedifferentiation of cells in embryo. In 1998, amethod was discovered to derive stem cells fromhuman embryos (human embryonic stem cells—hESCs) and grow the cells in the laboratory.These embryos were produced for reproductivepurposes through in vitro fertilization (IVF)procedures. Thus, pluripotent stem cells wereisolated from the murine embryo in 1981 andfrom human embryo in 1998. Now, these plu-ripotent embryonic stem cells can be culturedbeyond the Hayflick limit without changing theirphenotype and genotype and can be used as aninfinite source for in vitro production of all celltypes of our body or for the generation of chi-meric animals.
Another breakthrough came in 2006 whenconditions for genetically reprogramming ofsome specialized adult cells and converting theminto a stem cell-like state were identified byresearchers. This new type of stem cell wasnamed as induced pluripotent stem cells (iPSCs).In 2007, Mario R. Capecchi, Martin J. Evans,and Oliver Smithies were awarded the NoblePrize in Medicine/Physiology for their discoveryregarding the principles in introducing specificgene modifications in mice by the use ofembryonic stem cells. The collaboration of thesescientists initially aimed to repair defects in the‘hprt gene’. They identified and selected cellsthat have undergone homologous recombination,thereby eliminating the defective gene. Thesecells were then implanted into a surrogatemother who gave birth to a knockout mice strainthat was homozygous to the inert gene. In thisway, these scientists developed a method toreplace defected gene by homologous recombi-nation and ensure heritability of the geneticmaterial. After this major discovery the stem cellresearch got the new direction.
9.3 Properties
All stem cells regardless of their source havesome general properties that help in their iden-tification among the population of normal cells.These major properties include their capability
152 S. Kulshreshtha and P. Bhatnagar
of dividing and renewing themselves for longperiods, their unspecialized nature and theirability to give rise to specialized cell types.
9.3.1 Stem Cells are Unspecialized
The fundamental characteristic of a stem cell isits unspecialized structure without any tissuespecificity. Therefore, stem cells cannot performspecialized functions. However, unspecializedstem cells can be converted into specializedcells, including heart muscle cells, blood cells,or nerve cells that can perform special functions.All stem cells are characteristically of the samefamily type (lineage).
A starting population of stem cells that pro-liferates for many months in the laboratory canyield millions of cells. If the resulting cellscontinue to be unspecialized, like the parentstem cells, the cells are said to be capable oflong-term self-renewal. The exact controllingfactors and molecular events that allow stemcells to be unspecialized are still mystery toresearchers.
It has taken scientists many years of trial anderror to learn to grow stem cells in the laboratorywithout allowing them to differentiate into spe-cific cell types. Therefore, an important area ofresearch is focused on understanding the signalsin a mature organism that causes a stem cellpopulation to proliferate and remain unspecial-ized until the cells are needed for repair of aspecific tissue. The internal signals are con-trolled by genes of cells, which are interspersedacross long strands of DNA, and carry codedinstructions for all the structures and functionsof a cell. The process of differentiation of stemcells is complex and involves precise signals andthe regulation of gene expression.
9.3.2 Ability to Self-Renew
Stem cells have a special ability to divide andrenew themselves for extended periods of time.Stem cells repeatedly proliferate and produce
copies of them. Unlike mature cells, which arepermanently committed to their fate, stem cellscan both renew themselves as well as create newcells of whatever tissue they belong to (and othertissues). An initial population of stem cells canproduce millions of cells within months in alaboratory setting.
Embryonic stem cells are able to proliferatefor a year or even longer without ever devel-oping into specialized cells in contrast to adultcells. Researchers are contemplating on the dif-ferent aspects that regulate stem cell renewaland the cells’ proliferation and differentiation.Bone marrow stem cells, for example, are themost primitive cells in the marrow and fromthem various types of blood cells are descended.Bone marrow stem cell transfusions (or trans-plants) were originally given to replace varioustypes of blood cells.
To ensure self-renewal, stem cells undergotwo types of cell division. Symmetric divisiongives rise to two identical daughter cells, bothendowed with stem cell properties, whereasasymmetric division produces only one stem celland a progenitor cell with limited self-renewalpotential (Fig. 9.1). Progenitors can go throughseveral rounds of cell division before finallydifferentiating into a mature cell. It is believedthat the molecular distinction between symmet-ric and asymmetric divisions lies in differentialsegregation of cell membrane proteins (such asreceptors) between the daughter cells.
9.3.3 Ability to Give Riseto Specialized Cell
The ability of stem cells to give rise to special-ized cells is a crucial one. In this process ofdifferentiation, unspecialized stem cells producespecialized cells. It is believed that genes regu-late the internal signals that trigger this process.These genes carry the specific code or instruc-tions for all the parts and functions of a cell.External signals are those outside of the cell,which include chemicals released from othercells, physical connections with nearby cells,
9 Recent Advances in Stem Cell Research 153
and various other molecules in the surroundingarea. Currently, scientists are searching forsimilarities and differences between the signalsfrom one stem cell to another. Ultimately,answers will allow researchers to find ways tocontrol stem cell differentiation. With control,scientists can more easily grow cells and tissuesfor specific functions.
9.3.4 Plasticity of Stem Cells
Plasticity is an important property of stem cellswhich allows these cells to be committed togenerating a fixed range of progeny. When theyhave been relocated, to make other specializedsets of cells appropriate to their new niche, theycan switch. The evidence for stem cell plasticityin rodents and man was explained by Poulsomet al. (2002).
Adult stem cells are surprisingly flexible intheir differentiation repertoires due to the exis-tence of different pathways. Adult stem cellstypically generate the cell types of the tissue inwhich they reside. A blood-forming adult stemcell in the bone marrow, for example, normallygives rise to the many types of blood cells suchas red blood cells, white blood cells, and plate-lets. Until recently, it had been thought that ablood-forming cell in the bone marrow (hema-topoietic stem cell) could not give rise to thecells of a very different tissue, such as nervecells in the brain. However, a number ofexperiments over the last several years haveraised the possibility that stem cells from onetissue may give rise to cell types of a completelydifferent tissue. This phenomenon is known asplasticity.
9.4 Classification
Stem cells can be classified according to theirplasticity. Different types of stem cells vary intheir degree of plasticity or developmental ver-satility. The categories into which they fallinclude: the totipotent stem cell, pluripotentstem cell, multipotent stem cell, and oligopotentand nullipotent stem cells (Fig. 9.2).
9.4.1 Totipotent Stem Cells
‘‘Toti’’ comes from the Latin word meaning‘‘whole’’ or ‘‘Total’’, so totipotent stem cellshave total potential to develop into any cell typeor organ/tissues including extra embryonic tis-sues. Totipotent stem cells develop during sexualreproduction when male and female gametes fuseduring fertilization to form a zygote. The zygoteis totipotent because its cells can become anytype of cell and they have limitless replicativeabilities. After fertilization, zygote divides intoidentical totipotent cells, which can later developinto any of the three germ layers of a human, i.e.,endoderm, mesoderm, or ectoderm. This stage is
Fig. 9.1 Symmetric and asymmetric divisions (modifiedfrom http://stemcells.nih.gov/info/scireport/chapter1.asp)
154 S. Kulshreshtha and P. Bhatnagar
called morula stage (16-cell stage); the totipotentcells of morula start shifting toward pluripotentstem cells called blastocyst.
9.4.2 Pluripotent Stem Cells
Pluripotent stem cells are those cells which areable to form all the different types of cells in thebody. Specialization in pluripotent stem cells isminimal, and therefore they can develop intoalmost any type of cell. Pluripotent cells showthe formation of tumor-like mass called tera-toma. Teratoma formation is a hallmark of truepluripotency. It results from the aggressiveproliferation and differentiation of transplantedstem cells.
The best example of pluripotent stem cell isembryonic stem cell (ESC). ESCs seem to bemore flexible compared to adult stem cells,because they have the potential to produce every
cell type in the human body. ESCs are derivedfrom inner cell mass, called blastocyst, of 5–6-days-old embryo. After the fertilization of anegg by a sperm, blastocyst which is a tiny andhollow sphere of 30–34 cells develops generallyin 5 days that has potential to develop into acomplete human being. In normal development,the blastocyst implants in the wall of the uterusto become embryo and then develops into amature organism. Outer layer of cells initiatesthe formation of the placenta and the inner cellmass starts differentiating into the progressivelymore specialized cells of the body. During theearly stages of development, ESCs present inspecific part of embryo may develop into eye,blood, muscle, nerve, liver cells, etc. WhenESCs which may become liver, are isolated andtransferred to another section of the embryo, canbe developed into eye or blood. This shows thatdifferentiation of stem cell depends on theirposition in embryo.
ESCs can be induced to replicate in anundifferentiated state for very long periods oftime and then produce specialized cells onlyafter differentiation, which can be used fortherapeutic and transplantation purposes. How-ever, undifferentiated ESCs can form a teratomaand therefore, could not be used directly fortissue transplants. In an experiment, hESCs havebeen successfully differentiated into tissuesderived from the three germ layers (Cai et al.2007). This proves that hESCs can differentiateinto a broad spectrum of cell types in culture.
The differentiation of hESCs is efficient andhas advantages and disadvantages. The mainadvantages of hESCs are that these cells candevelop into most of the cells/tissues of the bodyand are easy to cultivate in laboratory. Thesecells have great potential for developing futuretherapies to cure diseases. The major disadvan-tage of hESCs is that blastocyst must bedestroyed when cells are removed. Moreover,egg donation is an important and serious issuewhich creates ethical problems.
Recently, iPSCs are developed which aregenetically altered adult stem cells. These cellsare induced or prompted in a laboratory to takethe characteristics of ESCs. Although iPS cells
Fig. 9.2 Hierarchy of stem cells during differentiationshowing that differential potential decreases and special-ization increases at each stage of development
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behave like ESCs and express some of the samegenes that are expressed normally in ESCs, theyare not exact duplicates of ESCs.
9.4.3 Multipotent Stem Cells
Multipotent stem cells have the ability to dif-ferentiate into a limited number of specializedcell types. Multipotent stem cells typicallydevelop into any cell of a particular group ortype. For example, bone marrow stem cells canproduce any type of blood cell. However, bonemarrow cells do not produce heart cells. Mes-enchymal stem cells (MSCs) are multipotentcells of bone marrow that have the ability todifferentiate into several types of specializedcells related to them. These stem cells give riseto cells that form specialized connective tissues,as well as cells that support the formation ofblood. Fetal stem cells, adult stem cells, andumbilical cord stem cells are examples of mul-tipotent cells.
Fetal stem cell is the main site of hemato-poiesis and is rich source of hematopoietic stemcells (HSCs) that leads to the generation ofmultiple cell types in blood. Tissues extractedfrom the fetal pancreas, when transplanted intodiabetic mice, are reported to stimulate insulinproduction. The possible reason of this is due toa true stem cell, a more mature progenitor cell,or to the presence of fully mature insulin-pro-ducing pancreatic islet cells themselves.
Primordial germ cells are multipotent cellsthat have been isolated from the gonadal ridge, astructure that arises at an early stage of the fetusthat will eventually develop into eggs or spermsin the adult. Germ cells can be cultured in vivoand have been shown to form multiple cell typesof the three embryonic tissue layers.
Adult stem cells or somatic stem cells areundifferentiated cells, found throughout the bodyafter embryonic development. These cells mul-tiply by cell division to replenish dying cells andregenerate damaged tissues. Adult stem cells arepresent in the depth of organs along with mil-lions of ordinary cells and restock some of the
body’s cells. These stem cells are tissue specificin their location and are derived from adult tis-sues such as bone marrow, blood, eye, brain,skeletal muscle, dental pulp, liver, skin, and thelining of the gastrointestinal tract and pancreas.Usually, they have very limited power of divi-sion due to which these can develop into thecells of only one particular tissue, thereforeconsidered as multipotent stem cells. In sometissues, these cells sustain turnover and repairthroughout life. For example, stem cells of skinwill give rise to new skin cells and thoseobtained from the brain can differentiate intoblood cells and muscle tissue.
Adult stem cells are not only difficult toidentify and purify, but also difficult to maintainin the undifferentiated state during growth inculture medium. Culturing of adult stems cells ina definitive medium is a high priority of stemcell research. The one important feature of stemcell is that these stem cells can be manipulatedeasily and effectively.
An adult stem cell is a completely committedstem cell. This concept is one of the excitingdiscoveries of last millennium. There is nowevidence that some of blood stem cells appar-ently are committed adult stem cells that are ableto become a stem cell in a different organ.Committed blood forming stem cell can producedifferent cells performing different functions.There are experiments performed for bonemarrow transplantation in rats with damagedlivers which help the liver to re-grow partiallywith bone marrow-derived stem cells. Bonemarrow-derived stem cells in muscle connectivetissue, satellite cells in adults, and stem cellmarker expressing cells located in muscle con-nective tissue can be derived from bone marrowin adulthood (Dreyfus et al. 2004).
9.4.4 Oligopotent Stem Cells
These stem cells have the ability to differentiateinto just a few types of cells. A lymphoid stemcell is an example of an oligopotent stem cell.This type of stem cell cannot develop into any
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type of blood cell as bone marrow stem cells cando. They only give rise to blood cells of thelymphatic system such as T and B cells.
9.4.5 Unipotent Stem Cells
These stem cells have unlimited reproductivecapabilities, but can only differentiate into asingle type of cell or tissue. Unipotent stem cellsare derived from multipotent stem cells andformed in adult tissue. Skin cells are one of themost prolific examples of unipotent stem cells.These cells must readily undergo cell division toreplace damaged cells.
9.5 Stem Cell Technology
A stem cell has the potential to develop into allkinds of body cells. This versatility of stem cellscan be used as an important tool to solve manykinds of problems in biology and medicine andtreatment of numerous diseases to save lives.With further research, scientists will hopefullybe able to understand more about the intriguingproperties of stem cells and can then find waysto isolate, cultivate, and manipulate stem cellsfor therapeutic value. This process is called stemcell technology (Fig. 9.3). Researches on stemcells provide advance knowledge about thedevelopment of organism from a single cell andreplacement of damaged cells with healthy cellsin adult organisms.
9.5.1 Sources of Stem Cell
Stem cells can be isolated from different sour-ces. These can be efficiently obtained fromimmature tissues possessing undifferentiatedcells such as embryonic cells (Fig. 9.4). Differ-ent populations of stem cells exist in embryo andare arranged hierarchically. This arrangementmakes the isolation of stem cells easy, differentESCs can thus be isolated from different stagesof developing organism such as morula, blasto-cyst, placenta, amniotic fluid, organ primordia,
and umbilical cord blood. The stem cells are alsopresent in the adult organisms revealing thenondisappearing of adult cells after birth. How-ever, these cells dilute among differentiated cellsand also change their properties in response tothe change of niche and new demands of theorganism. Tissue-specific stem cells can beharvested from different tissues and at differenttime-points: from children tissues, adult tissues,tissues of elderly people, and even postmortem.
9.5.1.1 Embryonic Stem Cell Sources
(a) In vitro fertilizationThe main potential source of blastocysts for
stem cell research is IVF clinics. On adminis-trating fertility drugs, stimulated ovaries startproducing multiple mature eggs. These eggs areretrieved by surgical procedure and all donatedeggs are fertilized by sperms in order to increasetheir chance of producing a viable blastocyst thatcan be used for implantation in the womb.However, all blastocysts are not implanted;remaining can be stored in freezers which can bea major source of ESCs for use in medicalresearch.
IVF facilitates the formation of blastocysts byfusion of sperm (specific genetic traits) witheggs of donor. This will help us in the study ofparticular diseases. However, the creation ofstem cells/blastocysts specifically for researchusing IVF is ethically problematic for somepeople, because it involves intentionally creatinga blastocyst that will never develop into a humanbeing.(b) Nuclear transfer
Another potential way to produce ESCs isnuclear transfer. Nuclear transfer can be done byinserting the nucleus of an already differentiatedadult cell into a donated enucleated egg. Due toinsertion of nucleus, enucleated egg becomesnucleated which is then stimulated to form ablastocyst. This blastocyst is a source of ESCwhich are copies or clones of adult cell due tothe presence of nucleus of adult stem cell. TheESCs created by nuclear transfer are geneticallymatched to the person needing a transplant,
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therefore shows the less chance of rejection byimmune system.
There is a misconception that nuclear transfertechnique is similar to reproductive cloning. Theuse of nuclear transfer to develop disease-specificstem cells can be called research cloning, and
the use of this technique for tissue transplants iscalled therapeutic cloning. These terms are dis-tinguished from reproductive cloning in whichthe intent is to implant a cloned embryo in afemale’s womb and allow it to develop fully intoan individual. This was the technique by which
Fig. 9.3 Stem cell technology for replacing the damaged cells of human body (modified fromhttp://www.stemcellsforhope.com)
Fig. 9.4 Different stagesof embryo development asa possible source ofstem cells (modified fromhttp://www.godandscience.org/slideshow/stem006.html)
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‘‘Dolly’’ the sheep was made and is now widelyused for reproductive cloning in animals. Inhumans, however, reproductive cloning has beendiscouraged by most of researchers.
Unfortunately, nuclear transfer has not beensuccessful in the production of hESCs. Scientistsmay use this technique to develop human stemcells in the future for studying the developmentand progression of specific diseases. Thismethod may be useful in creating the stem cellsthat contain only DNA of a patient sufferingfrom a specific disease.(c) Cord blood and aborted fetus
Recent researches have shown that ESCs canbe obtained from cord blood which may increasechances of the acceptance of stem cell research.Besides cord blood, stem cells can be harvestedfrom aborted fetus.(d) Amniotic fluid
In the womb, fetus is bathed with amnioticfluid which contains fetal cells including MSCspossessing ability to make a variety of tissues.Many pregnant women elect to have amnioticfluid drawn to test for chromosome defects, theprocedure known as amniocentesis. This fluid isnormally discarded after testing, but Dr DarioFauza, a surgeon at Children’s Hospital, Bostonand a member of the stem cell program, have beeninvestigating the idea of isolating MSCs andusing them to grow new tissues for babies whohave birth defects detected while they are still inthe womb, such as congenital diaphragmatichernia. These tissues would match the babygenetically, hence face less chances of rejectionby the immune system, and can be easilyimplanted either in utero or after the baby is born.
9.5.1.2 Adult Stem Cell Sources
1. Manipulating differentiated cellsSpecialized adult human cells can be repro-
grammed genetically by introducing specificgenes to create iPSCs (stem cell-like state). TheseiPSCs can be differentiated to any cell type.2. Somatic cells
Stem cells are thought to reside as quiescent(nondividing) cells in a specific area of each
tissue called a ‘‘stem cell niche’’. These cells arealso called somatic stem cells and begin todivide and create new cells only when they areactivated by tissue injury, disease, or anythingelse that makes the body need more cells. Manytissues have been reported to possess somaticstem cells such as bone marrow, blood vessels,and skeletal muscles.3. Other adult stem cell sources
Many researchers have reported the presenceof certain kind of adult stem cells which cantransform, or differentiate, into apparentlyunrelated cell types. This phenomenon is calledtransdifferentiation and has been reported insome animals. Differentiation of brain stem cellsinto blood cells and blood forming cells intocardiac muscle cells is the best example oftransdifferentiation process. Many questions areunanswered with this process such as how ver-satile they are, whether this is present in humanstem cells, can these be synthesized reliably inlaboratory. Stem cells are also present in tumorswhich are responsible for the growth of cancers.Hence, stem cells can be isolated from tumorwhich is removed from the body by surgicalprocedure.
Unlike ESCs, the use of adult stem cells inresearch and therapy is not controversial,because the production of adult stem cells doesnot require the creation or destruction of anembryo. However, the demand for autologous,patient-specific stem cells for regenerative ther-apies outstrip their supply. Several types of adultstem cells have been described which are givenbelow:(a) Hematopoietic stem cells
Blood stem cells have the ability to regener-ate all types of blood cells; therefore can recre-ate an entire new blood system. Stem cellsderived from bone marrow, when transplantedby intravenous injection to patients produce anentirely new blood system which helps thepatients in recovering from cancer. There is aroom for research that how and why stem cellsfind their way to the bone marrow site andregenerate a new blood system.
Recently, clinicians and scientists havefocused on CXCR4 as the protein that provides
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the ‘‘navigation system’’ to allow human bloodstem cells to find their way to the bone marrowand regenerate blood. The positive expressionfor CXCR4 has been widely hailed as a defini-tive marker for blood stem cells. Two distinctsubgroups of blood stem cells called CXCR4-positive and CXCR4-negative have been iden-tified. Their investigations showed that bothsubgroups possess the same blood cell regener-ation capabilities but show other unique func-tional and physiological features including howthey respond to growth factors. The two sub-groups also preferentially differentiate into dif-ferent types of blood cells: CXCR4-positive todisease fighting immune cells and CXCR4-neg-ative to red blood cells (http://www.robarts.ca/discovery-new-stem-cell-properties-advance).
HSCs are very rare, hence, their isolationand tracking in patients is difficult. Blood stemcells are present in bone marrow stromal stemcells (BMSCs) that have become a standard inthe field of adult stem cell biology and inregenerative medicine due to their high differ-entiation potentials and low morbidity duringharvesting. Nevertheless, harvesting of BMSCsby bone marrow aspiration is a painful proce-dure and the number of cells acquired is usu-ally low.(b) Mammary stem cells (MaSCs)
MaSCs provide cells for growth of themammary gland during puberty and gestationand play an important role in carcinogenesis ofthe breast. The mammary gland is a dynamicorgan undergoing significant developmentalchanges during puberty, pregnancy, lactation,and involution. Numerous studies have providedstrong evidence for the existence of MaSCsbased on the fact that the mammary gland can beregenerated by transplantation of epithelialfragments in mice. Shackleton et al. (2006)isolated discrete populations of mouse mam-mary cells on the basis of cell-surface markersand identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched forMaSCs. MaSCs have been isolated from human-and mouse- tissue as well as from cell linesderived from the mammary gland.
These stem cells capable of self-renewal anddifferentiation into the basal and luminal lin-eages comprise the functional mammary epi-thelium and have the ability to regenerate theentire organ in mice. The self-renewing capacityof these cells was demonstrated by serial trans-plantation of clonal outgrowths.
Recently in 2010, it was reported that MaSCsare highly responsive to steroid hormone sig-naling. However, they lack estrogen and pro-gesterone receptors. Ovariectomy markedlydiminished MaSC number and outgrowthpotential in vivo, whereas MaSC activityincreased in mice treated with estrogen plusprogesterone (Labat et al. 2010).(c) Mesenchymal stem cell
Mesenchymal stem cells (MSCs) are ofstromal origin and may differentiate into avariety of tissues/cells including chondrocytes,which produce cartilage. MSCs have been iso-lated from placenta, adipose tissue, lung, bonemarrow and blood, Wharton’s jelly from theumbilical cord and teeth (perivascular niche ofdental pulp and periodontal ligament).
MSCs cultured on regular flat surfaces suchas petridishes, rapidly lose their multipotentproperties which render them useless for stemcell experiments or clinical use. This problem issolved by using nanopatterned surfaces for theculturing of MSCs which helps in maintainingtheir phenotypic characteristics such as multi-potency over long culture periods (McMurrayet al. 2011). Generally, MSCs cultured withosteogenic media have very great efficiency ofproducing bone mineral in vitro. Similar effi-ciency can be achieved by the use of nanoscaledisorder to stimulate human MSCs even in theabsence of osteogenic supplements. However, itwas reported that topographically treated MSCshave a distinct differentiation profile comparedwith those treated with osteogenic media, whichhas implications for cell therapies. This showsthat stem cell differentiation to become osteo-blasts occurs in the absence of chemical treat-ment without compromising material properties.
MSCs are involved in osteoblast differentia-tion. Metabolic acidosis can alter osteoblast
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differentiation from MSCs through its variouseffects on osteoblastic genes. Furthermore,osteoblastic genes produce different proteinswhich result in an impairment of boneformation.
Defect in MSCs can be corrected by geneticmodification. After genetic modification, MSCscan be maintained in healthy and undifferenti-ated state. Furthermore, the genetically modifiedMSCs are able to engraft into many tissues ofunconditioned transgenic mice making them anattractive therapeutic tool in a wide range ofclinical applications. Chronic MSCs are alsoattractive for clinical therapy due to their abilityto differentiate, provide trophic support, andmodulate innate immune response. Researchersfrom Boston University’s School of DentalMedicine proved that bone marrow-derivedMSCs can relieve orofacial pain in rats withinone day of treatment by either intravenousinjection or direct injection of cells to the injuredarea.(d) Endothelial progenitor cells (EPCs)
Endothelial progenitor cell is a type of mul-tipotent adult stem cell found in the bone mar-row. These cells are found to line blood vesselsand originated from the hESCs. The differenti-ation of hESCs into endothelial cells avoids theformation of an embryoid-body intermediate.
Normal bone marrow sinusoidal endothelium,umbilical cord endothelium, and human telo-merase reverse transcriptase (hTERT)-immor-talized endothelial cell lines can support long-term proliferation and differentiation of humanstem cells. On transducing these cells with ad-enovectors expressing various hematopoieticgrowth factors (thrombopoietin; erythropoietin;granulocyte–macrophage colony-stimulatingfactor, GM-CSF; and c-kit Ligand, flt3/flk-2ligand), lineage appropriated expansion ofmature hematopoietic cells (red cells, megakar-yocytes, and neutrophils), as well as prolongedexpansion of pluripotent stem cells is observed.These transduced endothelial cells are used byresearchers for support of stem cell expansion ona clinical scale in a bioreactor system (http://www.mskcc.org/research/lab/malcolm-moore/lab/endothelial-cell-biology).
Bone marrow-derived endothelial stem cells(angioblasts) also exist in the circulatory system.In a study, at first dogs had been transplantedwith allogeneic marrow and further grafted withartificial arteries which rapidly became linedwith endothelium that was of the marrow donorgenotype. This formation of new blood vessels isknown as angiogenesis.
A rare population of cells comprising\1 % ofthe total CD34 positive population of human bonemarrow, mobilized blood, or umbilical cord bloodwere found to express the vascular endothelialgrowth factor (VEGF) Receptor-2 (VEGFR2),CD34, and the HSCs antigen AC133 (http://www.mskcc.org/research/lab/malcolm-moore/lab/endothelial-cell-biology). Purified CD34possessing hematopoietic progenitor cells, iso-lated from adults, can differentiate ex vivo to anendothelial phenotype. These cells were called asEPCs which showed expression of variousendothelial markers, and incorporated into neo-vessels at sites of ischemia. Hence, EPCs arepositive for both hematopoietic stem cell markers(CD34) and an endothelial marker protein(VEGFR2). However, CD34 is not an exclusivemarker of HSCs and found to be present also onmature endothelial cells. In later studies, CD133,also known as prominin, was found to be suitablemarker for immature hematopoietic stem cellwhich helps stem cells to differentiate in endo-thelial cells in vitro (Urbich and Dimmeler 2004).CD133 or AC133 is a highly conserved antigenwith unknown biological activity. It is notexpressed on mature endothelial cells andmonocytic cells but present on HSCs.(e) Neural stem cells
Neural stem cells exist not only in thedeveloping mammalian nervous system but alsoin the adult nervous system of all mammalianorganisms, including humans. The number ofviable locations for neural stem cell is limited inthe adult. Neural stem cells can also be derivedfrom primitive ESCs. These are thought to beexisted in the adult brain. The presence of stemcells in the mature primate brain was firstreported in 1967. The clue for this has beenfound by the process of neurogenesis, the birthof new neurons that continues to adulthood in
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rats. Normally, adult neurogenesis is restrictedto two areas of the brain—the subventricularzone, which lines the lateral ventricles, and thedentate gyrus of the hippocampus. However, thepresence of true self-renewing stem cells hasbeen debated. In ischemia, neurogenesis can beinduced in other parts of brain such as neocortexfollowing tissue damage. It has since beenshown that new neurons are generated in adultmice, songbirds and primates, and includinghumans.
In vitro cultivation of neural stem cells leadsto the formation of neurospheres which arefloating heterogeneous aggregates of cells,containing a large proportion of stem cells.Neural cells can be propagated for extendedperiods of time and differentiated into bothneuronal and glial cells and hence these areconsidered as stem cells. This behavior ofneural cell is induced by the culture conditionsin progenitor cells. The progeny of stem celldivision normally undergoes a strictly limitednumber of replication cycles in vivo. Further-more, neurosphere-derived cells do not behaveas stem cells when transplanted back into thebrain. Neurosphere-derived cells can differen-tiate into various cell types of the immunesystem if these are injected in blood. Potentialuses of these stem cells in repair includetransplantation to repair missing cells and theactivation of endogenous cells to provide ‘‘self-repair.’’(f) Olfactory adult stem cells
Olfactory stem cells have been successfullyharvested from the human olfactory mucosacells of the nose. These cells are also accessiblein humans from nasal biopsies. If the appropriatechemical environment is provided to these cells,they are able to act as multipotent type of stemcells which can develop into many different celltypes. Hence, they are easily grown in in vitroconditions and can be expanded to large num-bers and stored frozen.
Olfactory stem cells hold the potential fortherapeutic applications because these can beharvested with ease without harm to the patient.This means they can be easily obtained from allindividuals, including older patients who might
be most in need of stem cell therapies. Theolfactory neuroepithelium undergoes continualneurogenesis and after extensive lesions ordamage, fully regenerates to maintain sensoryfunction. The olfactory epithelium is one of thefew sites of adult neurogenesis, but the identityof its stem cell has been debated. Pathogens andother noxious substances constantly bombard theprimary olfactory receptor neurons in the nosewhich kills them and results in the loss of thesense of smell. After extensive injuries residentneuronal precursors are depleted. This results inthe loss of function of olfactory epithelium, ifthese neurons are not replaced. At this moment,horizontal basal cells (HBCs) transiently prolif-erate and their progeny fully reconstitutes theneuroepithelium. The pool of progenitor stemcells, that resides at the base of tissues, act assource of new neurons and can be used for thetreatment of diseases.(g) Testicular cells
Scientists at the School of Medicine, StanfordUniversity and at University of California, SanFrancisco have succeeded in isolating stem cellsfrom human testes. Previously, it was consideredas these cells show resemblance to ESCs andthey can differentiate into each of the three maintypes of tissues of the body (Science Daily2009). However, it is proved that the testes stemcells are not pluripotent and have different pat-terns of gene expression and regulation. Firstly,due to the expression of genes differ from ESCs,testicular stem cells expressed many, but not all,genes associated with pluripotency. Detailedanalysis of testicular stem cells reveals the dif-ferent pattern of methylation. Testicular stemcells show methylation at different region com-pared to ESCs which leads to the modification toDNA that affects gene expression. Secondly,they do not proliferate and differentiate asaggressively as hESCs. Finally, testicular stemcells have limited ability to form teratoma wheninjecting into immune-compromised mouse.These cells differ in gene expression, methyla-tion, and in their ability to form teratomas.Together, these results suggest that the stemcells isolated from male testes have some, butnot all the characteristics of true pluripotent
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cells. Hence, these testicular stem cells arecalled as multipotent stem cells.
These multipotent testicular stem cells havebeen derived from spermatogonial progenitorcells found in the testicles of laboratory mice.Similar types of cells were isolated from thetesticles of humans by the researchers of Ger-many and the United Kingdom. The extractedstem cells are known as human adult germlinestem cells (Waters 2008).(h) Umbilical cord blood stem cells
Recently, scientists have identified stem cellsin umbilical cord blood and the placenta ofnewborn baby that are multipotent in nature.These cells can give rise to the various types ofblood cells but not able to differentiate into allcell types of the body. These stem cells are foundto be genetically similar to the cells of newborn.Researchers at the University of Minnesotarecently found that they were able to reverse theeffects of stroke in lab rats using stem cells foundin human umbilical cord blood (http://news.nationalgeographic.com/news/2006/04/0406_060406_cord_blood.html). The transplantedstem cells took on properties of brain cells andseemed to be able to establish connection withbrain of rat. These experiments show the possi-bility of using these cells in the future for ther-apeutic purpose. However, extensive research isrequired in this field. Therefore, umbilical cordblood is often banked, or stored for future use.(i) Adipose stem cells
Adipose tissue is an attractive alternativesource of stem cells. Stem cells can be collectedin large quantities from adipose tissue frag-ments. Human adipose stem cells (ASCs) havetherapeutic applicability in pre-clinical studies indiverse fields, due to their ability to readilyexpand and their capacity to undergo adipo-genic, osteogenic, chondrogenic, neurogenic,and myogenic differentiation in vitro. Further-more, ASCs have been shown to be immuneprivileged and appear to be more geneticallystable in long-term culture compared to BMSCs.The safety and efficacy of ASCs for tissueregeneration or reconstruction is currently underassessment in clinical trials.
(j) Lung stem cellsLung stem cells include basal cells and
mucus-secreting cells in the trachea, Clara cellsin the bronchioles, and type II pneumocytes inthe alveoli. Mesenchymal cells predominate infetal lung development. In postnatal life, theClara cell is the most actively dividing cell in thetracheobronchial epithelium while type II pneu-mocytes are the progenitor cells for the alveolarepithelium. Under specific circumstances, basalcells and pulmonary neuroendocrine cells mayalso proliferate and act as stem cells.(k) Induced pluripotent stem cells
These are adult cells that have been geneti-cally reprogrammed and converted to an ESC-like state which can differentiate and formmultiple fetal cell types due to force to expressgenes and factors important for maintaining thedefining properties of ESCs. In animal studies,iPSCs have been shown to possess characteris-tics of pluripotent stem cells.
iPS and ESCs are very similar. Both can beused to grow nearly any cell type in the labunder precisely controlled conditions. They arealso self-renewing—they can divide and pro-duce their copies indefinitely. Both cell typescan help to understand how specialized celltypes develop from pluripotent cells. In thefuture, they might also provide an unlimitedsupply of replacement cells and tissues forpatients with many different diseases.
In contrast to ESCs, making iPS cells doesnot depend on the destruction of an earlyembryo. Some genes in iPS cells may behave ina slightly different way to those in ESCs. So atthe moment, ES cells cannot be replaced withiPS cells in basic research.
Reprogramming allows turning any cell ofthe body into a stem cell (Fig. 9.5). These werefirst developed for mouse in 2006 and for humanbeings in 2007. To develop these iPS cells,Yamanaka added four genes to skin cells iso-lated from a mouse. This leads to the initiationof reprogramming process which resulted in theconversion of these skin cells into iPSCs within2–3 weeks. Finally, in May 2008, the first iPScell line containing genetic disorder, a human
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sickle cell anemia iPS cell line, was successfullyestablished (Mali et al. 2008).
Moreover, this technique is used for repro-gramming of human cells by adding only threegenes. Mouse and human iPSCs possess impor-tant characteristics of pluripotent stem cells,including expressing stem cell markers, formingtumors containing cells from all three germlayers, and being able to contribute too manydifferent tissues. Currently, viruses are used tointroduce the reprogramming factors into adultcells (Fig. 9.6) which are found to cause cancers.Hence, this process must be carefully controlledand tested before the technique can lead touseful treatments for humans. Researchers arecurrently investigating nonviral delivery
strategies. Tissues derived from iPSCs will be anearly identical match to the cell donor, thusprobably avoid the rejection by the immunesystem. The iPSC strategy creates pluripotentstem cells that together with studies of othertypes of pluripotent stem cells will helpresearchers to know how to reprogram cells torepair damaged tissues in the human body.
Recently, iPS cell lines have established themodel for several diseases including adenosinedeaminase deficiency-related severe combinedimmunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome, Gaucher diseasetype III, Becker muscular dystrophy, Parkin-son’s disease (PD), Huntington’s disease, juve-nile-onset type 1 diabetes mellitus, Down
Fig. 9.5 Reprogramming of adult cells (modified from http://www.eurostemcell.org)
Fig. 9.6 Retroviral transfection of reprogramming genes
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syndrome/trisomy 21, and the carrier state ofLesch–Nyhan syndrome. The establishment of adiseased iPS cell bank would help to identifymechanisms for specific diseases and to discovereffective treatments (Zaehres and Scholer 2007;Mali et al. 2008; Park et al. 2008).
9.5.2 Isolation of Stem Cells
There are different approaches for the isolation ofstem cells largely depending on the tissue inwhich they are present. Generally, stem cells arepresent in the tissue which is made by these cells,e.g., bone stem cells can be taken from bonemarrow. MSCs, which can make bone, cartilage,fat, fibrous connective tissue, and cells that sup-port the formation of blood, can also be isolatedfrom bone marrow. The stem cell isolation isgreatly influenced by various factors dependingon the source and type of cells and the donor age.
9.5.2.1 Use of ImmunomagneticTechnique
The stem cells are diluted in the differentiatedcells of adults. The enrichment of stem cells inthe cell suspension by elimination of unwanteddifferentiated cells can be done rather than directstem cell isolation. This may be achieved by theuse of ferromagnetic nanoparticles labeled anti-bodies against the differentiated cells. Whenlabeled antibodies bind to the surface markers ofdifferentiated cells, the separation of these cellsoccur immunomagnetically. Sometimes, unla-beled antibodies are used against differentiatedcells and cells-antibodies complex are incubatedwith complement proteins for their lysis.
9.5.2.2 Removal of Factors SupportingGrowth of Differentiated Cells
Another method of isolation of stem cells isremoval of factors that supports survival of dif-ferentiated cells. For example, removal of pro-differentiation factors in serum and use of plasticdishes that do not support cell adhesion (helps inthe removal of differentiated cells). These
methods are often combined with those thatstimulate proliferation of stem cells while pre-serving their undifferentiated status, e.g., cellcultivation with the help of mitotic growthfactors.
9.5.2.3 Amendment of Stem CellsGrowth Supporting Factors
Since few stem cell-specific factors have alsobeen identified, these may be recognized byantibodies using fluorescence-activated cellsorter. Stem cell-specific factors permit propa-gation of stem cells in vitro and their expansion.Under stimulation with growth factors, stemcells may undergo symmetric divisions andincrease their numbers. As the growth of stemcells covers the dish in which they are cultured,replating them in a new culture dish is importantafter the cells reach a high density. The processof replating is called passaging.
9.5.2.4 Other Methods of IsolationAn interesting approach to enrich the stem cellsis gradient centrifugation against differentbuoyant densities. Stem cells have differentdensities than that of adult stem cells due tohaving large nucleocytoplasmic ratio. Therefore,density gradient centrifugation enriches thesecells in separators.
An interesting approach is to enrich stemcells as side population cells due to theircapacity to efflux Hoechst dye. This methodprovides a novel method of selection of trans-fected target cells possessing stem cell-associ-ated genes, which yields induced iPS cells.
9.5.3 Stem Cell Identification
9.5.3.1 Embryonic Stem CellsDuring the process of generation, stem cellsshould be assessed for fundamental properties.This process is called characterization. This canbe done by various methods at different stages ofstem cell growth. One of the most important
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properties of stem cells is their self-renewablecapability which can be assessed by growing andsubculturing stem cells for many months. Sub-culturing of stem cells also helps in determiningthe ability of revival of these after freezing.Healthy and undifferentiated cells as well as themorphology of chromosomes can be observedunder microscope. Presence of surface markersthat are found only on undifferentiated cells(Oct-4) can also be used for the identification ofstem cells. Marker Oct-4 is a transcription factorwhich turns genes on and off during the pro-cesses of cell differentiation and embryonicdevelopment.
The second important property of stem cellsis their pluripotency which can be tested byallowing the cells to differentiate spontaneouslyin cell culture and by manipulating the cells, sothat they will differentiate to form specific celltypes. Stem cells are able to form teratoma oninjecting the cells into an immunosuppressedmouse. This property of stem cells can be usedfor their identification.
9.5.3.2 Adult Stem CellsIn adults, stem cells are diluted amongst themature and differentiated cells, therefore, aredifficult to locate. In this case, the use of cell-specific molecular markers provides a goodeffective tool for locating the stem cells.Molecular markers are also applicable in label-ing the cells in a living tissue and then used fordetermination of the specialized cell types gen-erated by stem cells.
9.5.4 Culture of Stem Cells
The stem cells isolation is the first step in stemcell technology. Stem cells then need to begrown to large enough numbers to be useful fortreatment purposes. When the blastocyst is usedfor stem cell research, cells from inner cell massare isolated and placed in a culture dish with anutrient-rich liquid. Over the course of severaldays, the cells of the inner cell mass proliferate
and begin to crowd the culture dish. They are,therefore, removed gently and plated into sev-eral fresh culture dishes. This process is calledsubculturing and repeated many times for manymonths. After 6 months or more, the cells of theinner cell mass yield millions of ESCs. ESCsthat have proliferated in cell culture for 6 ormore months without differentiating are plurip-otent and appear genetically normal, are referredas an ESC line. Once cell lines are established,or even before that stage, batches of that can befrozen and shipped to other laboratories forfurther culture and experimentation. These ESCscan be used for therapeutic purposes also. To beuseful for producing medical therapies, culturedESCs will need to be differentiated into appro-priate tissues for transplantation into patients.Keeping this in view, researches are trying toachieve this type of differentiation. ES cell linesfrom patients with a variety of genetic diseasesprovide invaluable models to study thosediseases.
Sometimes, stem cell cultivation requires thepreparation of feeder layer in the culture dish forproviding support to growing stem cells. To coatthe culture dish with feeder layer, mouse skincells can be used which is treated in this waythat they will not divide after making a layer inthe inner surface of culture dish. Now, human ormouse embryonic cells, isolated from inner cellmass, are placed in this culture dish and nutrientmedium is poured over them. Feeder layer notonly provides a sticky surface for the inner cellmass to which they can attach but also releasenutrients into culture medium. However, culturemedia can be designed that does not requirefeeder layer.
9.5.5 Strategies for DifferentiatingHuman ESC into Three GermLayers
Human ESCs are an attractive cell source fortransplantation therapy, regenerative medicine,and tissue engineering. To realize potential ofstem cells, it is essential to control ESC
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differentiation and to direct the development ofthese cells along specific pathways. Basic sci-ence in the field of embryonic development,stem cell differentiation, and tissue engineeringhas offered important insights into key pathwaysand scaffolds that regulate hESCs differentiation,which have produced advances in modelinggastrulation in culture and in the efficientinduction of endoderm, mesoderm, ectoderm,and many of their downstream derivatives.
The basic methods of hESC differentiationare divided into three categories:1. Direct differentiation as a monolayer on
extracellular matrix proteins.2. Differentiation in co-culture with stromal
cells.3. The formation of 3-D spherical structures in
suspension culture, termed embryoid bodies.
9.6 Applications
Stem cells have potential in different areas ofhealth and medical research (Fig. 9.7). To startwith, studying stem cells will help to understandhow they transform into the dazzling array ofspecialized cells that make us what we are.Some of the most serious medical conditionssuch as, cancer and birth defects are due toproblems that occur somewhere in this process.A better understanding of normal cell develop-ment will allow us to understand and perhaps
correct the errors that cause these medicalconditions.
Diseases treated by these nonembryonic stemcells include a number of blood and immune-system related genetic diseases, cancers, disor-ders, juvenile diabetes, Parkinson’s, blindness,and spinal cord injuries. However, the use of thistechnology for treating various diseases is notvery easy because of several problems associ-ated with it. Besides the ethical problems ofstem cell therapy, there is a technical problem ofgraft-versus-host disease associated with allo-geneic stem cell transplantation. However, theseproblems associated with histocompatibilitymay be solved using autologous donor adultstem cells or via therapeutic cloning.
9.6.1 Therapy for Cancer
Cell therapy has the potential to offer noveltreatment modalities for a number of diseasesincluding cancer. MSCs play a particularlyimportant role in the regulation of both solidtumor and hematological malignancies. MSCshave tumor-homing properties which may beimportant to use them as targeted delivery vehi-cles of gene products. However, conflictingreports have found that human MSCs can pro-mote metastasis, while on the other hand otherstudies have shown that MSCs can stall thegrowth of metastatic lesions. About half of the
Fig. 9.7 Stem cellresearch in toxicology,drug design and geneticcontrol (modified fromhttp://science.jburroughs.org/mbahe/BioEthics/ppt_pdf/030%20StemCells.pdf)
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tumor-burdened animals that were treated withmurine and human MSCs, respectively harboredmetastatic nodules significantly higher thancontrols (17 %) showing metastatic nodules.Hence, both human and mouse MSCs possessmetastasis-promoting activity raising concernsabout the safe use of MSCs. Although at the sametime efforts are going on to make use of murinetransgenic model systems feasible to study therole of MSCs in metastasis development andpossibly finding ways of using them safely as celltherapeutic vehicles (Albarenque et al. 2011).
One can examine methods to take advantageof the unique immune properties of MSCs toimprove therapy for cancer. Targeting MSCsmight be more of a challenge due to the cellsendogenous functions. MSCs have immunesuppressive power which might partly explainreduced enthusiastic outcome of immune ther-apy for tumor.
9.6.2 Therapy for Cystic Fibrosis
The heterogeneous stem cell populations arepresent in bone marrow and lung tissue. Theirpresence in lung tissue may lead to stem cellmanipulative and regenerative therapies forconditions such as idiopathic pulmonary fibrosisand cystic fibrosis transmembrane regulatorgene therapy for cystic fibrosis.
9.6.3 Stem Cells in Tissue Repair
Organ-specific progenitors and HSCs areinvolved in repair of tissues. Epidermal stemcells and bone marrow progenitor cells bothcontribute for wound healing in the skin. Tissueregeneration is achieved by two mechanisms: (1)circulating stem cells divide and differentiateunder appropriate signaling by cytokines andgrowth factors, e.g., blood cells; and (2) differ-entiated cells which are capable of division canalso self-repair, e.g., hepatocytes, endothelialcells, smooth muscle cells, keratinocytes, andfibroblasts. These fully differentiated cells are
limited to local repair. For more extensiverepair, stem cells are maintained in the quiescentstate, and can then be activated and mobilized tothe required site.
Adult stem cells have shown promise as a cellsource for a variety of tissue engineering andcell therapy applications. MSCs secrete a largespectrum of bioactive molecules. These mole-cules are immunosuppressive, especially for T-cells, thus allogeneic MSCs can be consideredfor therapeutic use. In this context, the secretedbioactive molecules provide a regenerativemicroenvironment for a variety of injured adulttissues to limit the area of damage and to mounta self-regulated regenerative response. Thisregenerative microenvironment is referred to astrophic activity; therefore, MSCs appear to bevaluable mediators for tissue repair and regen-eration. The natural titers of MSCs that aredrawn to sites of tissue injury can be augmentedby allogeneic MSCs delivered via bloodstream.Indeed, human clinical trials are now under wayto use allogeneic MSCs for treatment of myo-cardial infarcts, graft-versus-host disease, Cro-hn’s disease, cartilage and meniscus repair,stroke and spinal cord injury.
Recent reports indicate that umbilical cordblood stem cells may be beneficial for spinalcord injury and that lithium may promoteregeneration and recovery of function afterspinal cord injury. Both lithium and umbilicalcord blood are widely available therapies thathave long been used to treat diseases in humans.
9.6.4 Intervertebral DisKRegeneration
In humans, lower back pain is one of the mostcommon causes of morbidity. In the normalintervertebral disk, the nucleus pulposus exerts ahydrostatic pressure against the constrainingannulus fibrosus, which allows the disk tomaintain flexibility between adjacent vertebrae,while absorbing necessary compressive forces.The nucleus pulposus performs this role becauseof its hydrophilic gel-like structure. The
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extracellular matrix of the nucleus pulposus isup to 80 % hydrated, as a result of large amountsof the aggregating proteoglycan, chondroitinsulfate proteoglycan. Intervertebral disk degen-eration (IDD) is characterized by a progressivedecrease in proteoglycan content and cell den-sity in the nucleus pulposus.
Current therapies for IDD are aimed attreating the clinical symptoms arising from IDDrather than directly treating the underlyingproblem. Different strategies using stem cells tosupplement/replenish the disk cell populationhave been proposed to slow, stop, or reverse theprogressive loss of proteoglycan. Paracrineeffects, cell–cell interactions are importantcharacteristics that make stem cells as a sourceof cells for intervertebral disk (IVD) tissueengineering and cell therapy. Stem cell helps inthe supplementation/replenishment of interver-tebral disk cells which further leads to synthesis/maintenance of a more functional ECM (extracellular matrix) in a degenerated disk. Trans-planted stem cells survive and successfullyengraft into the IVD tissue and are effectivevehicles for exogenous gene delivery to the IVD.Thus, stem cells might be able to confer thera-peutic effects in stem cell therapy of IVDdegeneration.
Adult stem cells can be derived from muscletissue. Muscle tissues serve as a good source ofstem cells because of its vast abundancethroughout the human body. Vadalà et al. (2008)reported a synergistic effect between musclesderived stem cells and NP cells resulting in anupregulated proteoglycan synthesis and NPCsproliferation in vitro.
Murrell et al. (2009) reported that olfactorystem cells could also differentiate into a nucleuspulposus chondrocyte phenotype in vitro and invivo after transplantation into the injured inter-vertebral disk. This success provided a rationalefor moving toward more extensive larger animalstudies for assessment of regeneration and reliefof symptoms. Chen et al. (2009) focused on theneed to evaluate the in vivo effect on synovial-derived stem cells on disk regeneration.
9.6.5 Functional Genomic Studies
The ability to manipulate a specific gene of EScells of mouse has been demonstrated sincelong. Mouse ES cells are able to producetransgenic animals. The study of the function ofmammalian genes and proteins in the mouse wasreported by Floss and Wurst (2002).
Stem cells help in the study of functional genesand the replacement of defective genes with thefunctional ones. Pelizaeus-Merzbacher disease(PMD) patients and myelin-deficient (md) ratscarry mutations in the X-linked gene encodingmyelin proteolipid protein (PLP). ES cell-derivedoligodendrocytes can form myelin in vivo. Thecells were grown in the presence of fibroblastgrowth factor (FGF2) and platelet-derivedgrowth factor (PDGF) and then transplanted intothe spinal cord of 1-week-old md rats. Two weeksafter transplantation of ES cell-derived precur-sors into the dorsal columns of the spinal cord,numerous myelin sheaths were found in six ofnine affected md-male rats. This experimentproposed the replacement of the defective genewith functional gene. It will also help in studyingrole of functional gene in an organism.
9.6.6 Study of Biological Processes
Studies of biological processes namely develop-ment of the organism is facilitated by the ability totrace stem cell fate. The spleen colony assay canbe used in the study of the development of bloodcells. In this method, single cells are injected intoheavily irradiated mice so that all the hemato-poietic cells are destroyed and new blood cells inthese mice originate from the original colony.
9.6.7 Drug Discoveryand Development
The isolation and purification of mouse ES cellsand genetic engineering techniques have led tothe use of murine ES cells in drug discovery.
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With the sequencing of the human genome manypotential targets of new drugs have beenidentified.
Normal and disease-specific iPS cell lineswill prove extremely useful in studying how adrug influences a disease. BMSCs have becomea standard in the field of adult stem cell biologyand in regenerative medicine due to their highdifferentiation potentials and low morbidityduring harvesting.
9.6.8 Stem Cells in Liver Disease
A number of animal and human studies dem-onstrated that either HSCs or MSCs could beapplied to therapeutic purposes in certain liverdiseases. The diseased liver may recruit migra-tory stem cells, particularly from the bone mar-row, to generate hepatocyte-like cells either bytransdifferentiation or cell fusion. Transplanta-tion of BMSCs has therapeutic effects of resto-ration of liver mass and function, alleviation offibrosis, and correction of inherited liver dis-eases. Several potential approaches for BMSCsdelivery in liver diseases have been proposed inanimal studies and human trials. BMSCs can bedelivered via intra-portal vein, systemic infu-sion, intraperitoneal, intrahepatic, and intra-splenic. The optimal stem cell delivery shouldbe easy to perform, less invasive and traumatic,with minimum side effects, and high cells sur-vival rate (Xu and Liu 2008).
BMSCs were transplanted into liver-injuredrats to test the therapeutic effect. BMSCsexpressed the albumin mRNA and production ofprotein after cultivation with hepatocyte growthfactor (HGF) for 2 weeks. The BMSCs appearedto differentiate into hepatocyte-like cells whichwere transplanted into carbon tetrachloride(CCl4)-injured rats by injection through thecaudal vein. Transplantation of the BMSCs intoliver-injured rats restored their serum albuminlevel and suppressed transaminase activity andliver fibrosis. Therefore, BMSCs were shown tohave a therapeutic effect on liver injury (Yagiet al. 2008). MSCs were more resistant to
reactive oxygen species in vitro, reduced oxi-dative stress in recipient mice, and acceleratedrepopulation of hepatocytes after liver damage.
Hepatic progenitor cells from MSC differen-tiation in the growth-factor-free co-culture sys-tem may also contribute to the therapeutic effectfor liver diseases in vivo. MSCs from bonemarrow of green fluorescent protein-transgenicSprague–Dawley rats were co-cultured withhepatocytes from normal Sprague–Dawley rats,sharing growth-factor-free media. After 14 days,cells were implanted into the spleen of CCl4-injured rats and remarkably decreased fibrosiswas observed. CCl4/co-cultured MSC hadreduced alpha-fetoprotein expression, CK18,and CK19 exhibited stronger expression. Albu-min in CCl4/co-cultured MSC increased in pro-tein level (Li et al. 2010). Kuo et al. (2008)reported that both MSC-derived hepatocytes andMSCs, transplanted by either intrasplenic orintravenous route, engrafted recipient liver, dif-ferentiated into functional hepatocytes, and res-cued liver failure. Intravenous transplantationwas more effective in rescuing liver failure thanintrasplenic transplantation. In this type oftransplantation, paracrine effect was found to beeffective.
Hepatic cirrhosis is the end-stage of chronicliver diseases. The majority of patients withhepatic cirrhosis die from life-threatening com-plications occurring at their earlier ages. Thetransplantation of autologous bone marrow-derived mesenchymal stem cells holds greatpotential for treating hepatic cirrhosis. MSCscan differentiate to hepatocytes, stimulate theregeneration of endogenous parenchymal cells,and enhance fibrous matrix degradation. Exper-imental and clinical studies have shown prom-ising beneficial effects.
9.6.9 Acute Myocardial Infarction
A number of stem cell types, including ES cells,cardiac stem cells that naturally reside within theheart, myoblasts (muscle stem cells), adult bonemarrow-derived cells including mesenchymal
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cells (bone marrow-derived cells that give rise totissues such as muscle, bone, tendon, ligament,and adipose tissue), EPCs (cells that give rise tothe endothelium, the interior lining of bloodvessels), and umbilical cord blood cells, havebeen investigated as possible sources for regen-erating damaged heart tissue.
Stem cells that are injected into the circula-tion or directly into the injured heart tissueappear to improve cardiac function and/orinduce the formation of new capillaries. Themechanism for this repair remains controversialand the stem cells likely regenerate heart tissuethrough several pathways. Whether these cellscan generate heart muscle cells or stimulate thegrowth of new blood vessels that repopulate theheart tissue, or help via some other mechanismis actively under investigation. For example,injected cells may accomplish repair by secret-ing growth factors, rather than actually incor-porating into the heart.
By flow cytometry, myogenic stem cellpopulations expressing the neural cell adhesionmolecule can be selected in the presence andabsence of a6 integrin. The expression of a6integrin showed an advantage in the formationof myotubes, possibly by an improved cellfusion capacity. It was possible to generatehealthy heart muscle cells in the laboratory andthen transplant those cells into patients withchronic heart disease. Preliminary research inmice and other animals indicated that BMSCs,transplanted into a damaged heart, can havebeneficial effects (Wilschut et al. 2011).
Recent randomized clinical trials indicate thatintracoronary delivery of bone marrow (stem)cells leads to an improvement in systolic func-tion after acute myocardial infarction, therebyproviding the first evidence that cell therapy maywork in patients (Wollert 2008).
Dual stem cell therapy comprises granulocytecolony stimulating factor (GCSF)-based stemcell mobilization and diprotin A mediateddipeptidyl peptidase-IV (DPP-IV) inhibition.Dual stem cell treatment effectively stimulatedthe pool of resident cardiac stem cells which was
reversed by AMD3100 treatment. According toTheiss et al. (2011), homing through the SDF-1/CXCR-4 axis is essential for the success of dualstem cell therapy.
Although adult stem cells with the capacity totransform into various cardiac cell types and tosecrete cardioprotective cytokines have beenidentified, endogenous repair mechanisms in theadult heart are not sufficient for meaningful tis-sue regeneration.
9.6.10 Stem Cell Therapy for Diabetes
In type 1 diabetes mellitus (T1DM), the cells ofthe pancreas that normally produce insulin aredestroyed by the patient’s own immune system.Any potential stem cell-based cure for T1DMshould address the need for beta-cell replace-ment, as well as control of the autoimmuneresponse to cells which express insulin.
It may be possible to direct the differentiationof hESCs in cell culture to form insulin-pro-ducing cells that eventually could be used intransplantation therapy for persons with diabe-tes. These stem cells reconstitute a functionalbeta-cell mass that have molecular characteris-tics closely resembling bonafides insulin-secreting cells. These cells express insulin;however, these cells are often unresponsive toglucose. The use of mesenchymal stromal cellsor umbilical cord blood to modulate the immuneresponse is already in clinical trials; however,definitive results are still pending.
Ductal structures of the adult pancreas con-tain stem cells that differentiate into islets ofLangerhans. Here, pancreatic ductal epithelialcells isolated from prediabetic adult nonobesediabetic mice were grown in long-term cultures.They were induced to produce functioning isletscontaining cells. These in vitro generated isletsshowed temporal changes in mRNA transcriptsfor islet cell-associated differentiation markers,responded to in vitro glucose challenge andreversed insulin-dependent diabetes after beingimplanted into diabetic nonobese diabetic mice
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(Ramiya et al. 2000). The ability to controlgrowth and differentiation of islet stem cellsprovides an abundant islet source for cellreconstitution in type I diabetes. Recent successin islet transplantation-based therapies for dia-betes mellitus and the extreme shortage of pan-creatic islets have motivated recent efforts todevelop renewable sources of stem cells forislet-replacement tissue.
Increase in endothelial cell sloughing anddiminished function of endothelial stem cellprogenitors in diabetic subjects are well-knownphenomena. The transplantation of BMSCsincluding MSCs into type 2 diabetic mice wouldrestore insulin sensitivity and glucose tolerance.This approach, when combined with inductionof HO-1 (a cytoprotective antioxidant system) inthe recipient, would further improve bone mar-row function. BMSC administered by the intra-bone marrow–bone marrow transplantation(IBM–BMT) significantly increased BMSCfunction, serum adiponectin, and glucose toler-ance. It has been observed that induction of HO-1 in the recipients greatly enhanced the ability ofBMSC to prevent diabetes.
ESC can be differentiated into insulin-producing cells by manipulating culture condi-tions. In vitro differentiation of mouse ESC cangenerate embryoid bodies, which, after selectionfor nestin expressing ESC, were stimulated todifferentiate toward different cell-like pheno-type. For example, the addition of phosphoino-sitide kinase inhibitors promoted differentiationof larger numbers of ESC toward functionalcells (Hussain and Theise 2004).
Pancreatic endocrine precursor cells onbasement membrane extract (BME) differentiateinto insulin-producing cells. A thin BME coatingis sufficient to maintain an undifferentiatedphenotype during embryonic stem cell expan-sion, while a thick BME hydrogel may beemployed to induce stem cell differentiation.Stem cell differentiation in vitro can be used toidentify cell types based on morphology andgene expression, and also to determine potentialfunctionality in vivo (Arnaoutova et al. 2012).
9.6.11 Therapy for Parkinson’s Disease
Parkinson’s disease (PD) is a neurodegenerativedisorder, symptomatized by tremors, rigidity,bradykinesia etc. In this disease, the majority ofthe signs and symptoms appear because of theprogressive loss of cells in a small area known asthe substantia nigra. These cells make dopa-mine, which is delivered to a part of the basalganglia known as the striatum; when nigralneurons die and striatal dopamine diminishes,the signs and symptoms of Parkinson’s diseasebecome manifested. Thus, replenishing missingneurons in a limited area of the brain should intheory reverse parkinsonism, making this anattractive approach. But the challenge of actu-ally replacing injured and/or lost neurons in theadult human nervous system has proven to be adaunting task. Stem cells might be effective incase of different forms and at all stages ofparkinsonism.
Adult stem cells harvested from the noses ofParkinson’s patients gave rise to dopamine-pro-ducing brain cells (http://www3.griffith.edu.au/03/ertiki/tiki-read_article.php?articleId=16841). When these stem cells were culturedand injected into the damaged area the rats re-acquired the ability to run in a straight line. Stemcells from the olfactory nerve in the nose are‘naive’, having not yet differentiated into whichsort of cells they will give rise to. They can stillbe influenced by the environment they are putinto.
Dopaminergic neurons can also be obtainedfrom fetal brain tissue. At stages I–III, when uni-and bi-lateral manifestations of the disease areobserved, such as balance disturbances, tremor,mild and moderate rigidity etc., administrationof fetal stem cells is effective in 85 % of cases.After the treatment, patients report improvementof stiffness, decrease in shaking while walkingand tremor. The dose of antiparkinson drugs canbe lowered. At stage IV (severe stiffness, butthe patient still can walk or stand unassisted),stem cell treatment is effective in 65–70 %of Parkinson’s disease cases. At stage V
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(bed- or wheelchair-bound), this therapy helps toimprove patient’s quality of life and ease carefor the patient due to decreased spasticity andtremor syndrome and improved sleep, normali-zation of organ, and system functioning (heart,lungs, and bowels). In 100 % of cases of PD,treatment with fetal stem cells results in psy-choemotional improvements. After the stem celltreatment, patients demonstrate positive emo-tions, improved thinking, more expressive,intelligible and louder speech, and more persis-tent memory and intellect. These trials provideproof of principle for the approach, since in afew of these trials major and long lastingimprovements were seen in some patients. Thetrials also emphasized several issues that need tobe resolved, one of which is the need to producelarge amounts of pure, uniform cells for trans-plantation into patients. Recent findings alsohighlight a further concern about cell trans-plantation therapies. The fetal transplants thatsome patients received began to show signs ofbeing affected by Parkinson’s disease. Thisshowed that the disease from the patient wastransmitted to the transplanted fetal cells(http://www.eurostemcell.org/).
Mouse iPS cell-derived neurons could func-tionally integrate into donor brains, and symp-toms of Parkinson’s disease in donor mice wererelieved after transplantation of iPS cell-deriveddopaminergic neurons (Wernig et al. 2008).
Human-induced pluripotent stem cells (hiP-SCs) are a potentially unlimited source ofpatient-specific cells for transplantation. Dopa-mine (DA) neurons derived from protein-basedhiPSCs exhibited gene expression, physiological,and electrophysiological properties similar tothose of midbrain dopamine (mDA) neurons.Transplantation of these cells into rats with stri-atal lesions, a model of PD, significantly rescuedmotor deficits. These data support the clinicalpotential of protein-based hiPSCs for personal-ized cell therapy of PD. However, it is critical toevaluate the safety of hiPSCs generated by dif-ferent reprogramming methods. On comparingmultiple hiPSC lines derived by virus- and pro-tein-based reprogramming to hESCs, it wasfound that neuronal precursor cells (NPCs) and
DA neurons delivered from lentivirus-basedhiPSCs exhibited residual expression of exoge-nous reprogramming genes, but those cellsderived from retrovirus- and protein-based hiP-SCs did not. Furthermore, NPCs derived fromvirus-based hiPSCs exhibited early senescenceand apoptotic cell death during passaging, whichwas preceded by abrupt induction of p53. Incontrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable withoutsenescence (Rhee et al. 2011). The use of virusesencoding the reprogramming factors represents amajor limitation of the current technology sinceeven low vector expression may alter the differ-entiation potential of the hiPSCs or inducemalignant transformation. Soldner et al. (2009)reported that residual transgene expression invirus-carrying hiPSCs can affect their molecularcharacteristics and that factor-free hiPSCs,therefore represent a more suitable source ofcells for modeling of human disease.
9.6.12 Therapy for Bone and CartilageRepair
Bone and cartilage defects are common featuresof bone fracture and joint diseases, such asrheumatoid arthritis or osteoarthritis that havegreat social and economic impact on the agingoccidental population. Injured cartilage tissuecannot spontaneously heal and, if not treated,can lead to osteoarthritis of the affected joints.Although a variety of procedures are beingemployed to repair cartilage damage, methodsthat result in consistent durable repair tissue arenot yet available. Tissue engineering is arecently developed science that merges the fieldsof cell biology, engineering, material science,and surgery to regenerate new functional tissue.Three critical components in tissue engineeringof cartilage are as follows: First, sufficient cellnumbers within the defect, such as chondrocytesor multipotent stem cells capable of differenti-ating into chondrocytes; second, access togrowth and differentiation factors that modulatethese cells to differentiate through the chondro-genic lineage; third, a cell carrier or matrix that
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fills the defect, delivers the appropriate cells, andsupports cell proliferation and differentiation(Gao et al. 2007). Stem cells that exist in theembryo or in adult somatic tissues are able torenew themselves through cell division withoutchanging their phenotype and are able to dif-ferentiate into multiple lineages including thechondrogenic lineage under certain physiologi-cal or experimental conditions.
Recent investigations on the stromal MSCoffer a new perspective for bone and cartilagetissue engineering. Xian and Foster (2006) pro-posed few points for exploring the potentials ofMSCs in cartilage repair. These include (a)identifying readily available sources of anddevising appropriate techniques for isolation andculture expansion of MSCs that have goodchondrogenic differentiation capability; (b) dis-covering appropriate growth factors (such asTGF-beta, IGF-I, BMPs, and FGF-2) that pro-mote MSC chondrogenic differentiation; (c)identifying or engineering biological or artificialmatrix scaffolds as carriers for MSCs and growthfactors for their transplantation and defect fill-ing. Richardson et al. (2010) exposed implantedMSCs to traumatic physical loads and highlevels of locally produced inflammatory media-tors and catabolic cytokines. They also exploredthe potential of culture models of MSCs, fullydifferentiated cells and co-cultures as ‘‘proof ofprinciple’’ ethically acceptable ‘‘3Rs’’ modelsfor engineering articular cartilage.
Caplan (2005) used MSCs with site-specificdelivery vehicles to repair cartilage, bone, ten-don, marrow stroma, muscle, and other con-nective tissues. In the beginning of the twenty-first century, they have made substantialadvances: the most important is the developmentof a cell-coating technology, called painting thatallows us to introduce informational proteins tothe outer surface of cells. These paints can serveas targeting addresses to specifically dock MSCsor other reparative cells to unique tissueaddresses. The scientific and clinical challengeinclude to perfect cell-based tissue engineeringprotocols to utilize the body’s own rejuvenationcapabilities by managing surgical implantations
of scaffolds, bioactive factors, and reparativecells to regenerate damaged or diseased skeletaltissues. Arinzeh (2005) evaluated the efficacy ofMSC-loaded scaffolds in large bone defects as apotential substitute for autologous and alloge-neic bone grafts.
Current strategies to engineer cartilage tissuesfrom adult stem cells and human ESC-derivedcells are also in developing state (Hwang andElisseeff 2009). Earlier, Hwang et al. (2007)demonstrated that ESCs encapsulated withinpolyethylene glycol-based photopolymerizinghydrogels and cultured in an appropriate growthfactor and medium conditions undergo chon-drogenic differentiation with extracellular matrixdeposition characteristic of neocartilage. Vinatieret al. (2009) evaluated the optimal combinationsthat will answer to the functional demand placedupon cartilage tissue replacement in animalmodels and in clinics.
Similar to those of bone marrow-derivedmesenchymal stem cells (BMSCs), adipose-derived adult stem (ADAS) cells show charac-teristics of multipotent adult stem cells andunder appropriate culture conditions, synthesizecartilage-specific matrix proteins that areassembled in a cartilaginous extracellularmatrix. The growth and chondrogenic differen-tiation of ADAS cells is strongly influenced byfactors in the biochemical as well as biophysicalenvironment of the cells.
Autologous chondrocyte transplantation(ACT) is technically feasible and biologicallyrelevant to repairing disk damage and retardingdisk degeneration. In ACT, a cartilage biopsy istaken from the patient and articular chondro-cytes are isolated. The cells are then expandedafter several passages in vitro and used to fill thecartilage defect. A dog model was used toinvestigate the hypothesis that autologous diskchondrocytes can be used to repair damagedintervertebral disk. Disk chondrocytes wereharvested and expanded in culture under con-trolled and defined conditions, returned to thesame animals from which they had been sam-pled (autologous transplantation) via percutane-ous delivery. The animals were analyzed at
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specific times after transplantation by severalmethods to examine whether disk chondrocytesintegrated with the surrounding tissue, producedthe appropriate intervertebral disk extracellularmatrix, and might provide a formative solutionto disk repair. Meisel et al. (2007) designedEuroDISC according to the German Drug Law(Arzneimittelgesetz) and good manufacturingpractices. This has been certified according tointernationally approved DIN EN ISO 9001standards. To broaden the scope, better under-standing and long-term efficacy, clinical trialwas initiated to assess the potential autologousdisk chondrocyte transplantation in a broaderpopulation. The clinical goals of the EuroDISC,provided satisfactory results such as long-termpain relief, maintain disk height, and preventadjacent segment disease.
Despite promising experimental approaches,however, none of the current cartilage repairstrategies have generated long-lasting hyalinecartilage replacement tissue that meets thefunctional demands placed upon this tissue invivo. The reasons for this are diverse and canultimately result in matrix degradation, differ-entiation or integration insufficiencies, or loss ofthe transplanted cells and tissues.
9.6.13 Stem Cells for Usein Regenerative Medicine
Stem cells are ideal candidates for use inregenerative medicine because of their ability toself-renew and to commit to multiple cell lin-eages. Stem cells for regenerative medicineapplications should meet the following criteria:1. These should be found in abundant numbers
(millions to billions of cells).2. These should be harvested by a minimally
invasive procedure with minimal morbidity.3. These should be differentiated along multiple
cell lineage pathways in a controllable andreproducible manner.
4. These should be safely and effectivelytransplanted to either an autologous or allo-geneic host.
5. These should be produced in accordance withcurrent good manufacturing practiceguidelines.Recent advancements in tissue engineering
and regenerative medicine have highlightedMSCs as a potential source of cells which woulddifferentiate to a variety of tissue tailored toindividual needs. Transplanted hMSCs have thepotential to migrate into normal and injured liverparenchyma, particularly under conditions ofchronic injury, but differentiation into hepatocyte-like cells is a rare event (di Bonzo et al. 2008).
9.6.14 Treatment of ImmunologicalDisorders and Other Diseases
MSC can migrate to injured tissues and some oftheir reparative properties are mediated by par-acrine mechanisms including their immune-modulatory actions. ASCs are good candidatefor cell-based therapies, because these can sur-vive in a low oxygen environment and secreteangiogenic cytokines. These cells also possessimmunomodulatory properties and protectiveeffects in immunological disorders. Reports haveshown that the immunosuppressive capacity ofthe ASCs may in some cases favor the growth oftumor cells but contradictory results exist andthe question is a matter of intense debate.
According to Lindroos et al. (2010), ASCsexert profound immune-modulatory propertiesand protective effects on acute graft-versus-hostdisease (GvHD) and experimental arthritis. Byfunctional characterization, ASCs have beenshown to be immune-privileged due to lack ofHLA-DR expression (major histocompatibilitycomplex class II) and the suppression of prolif-eration of activated allogeneic lymphocytes.Additionally, ASCs have been shown to inhibitthe production of inflammatory cytokines byboth CD4+ T helper cells and CD8+ T cytotoxiccells, stimulate the production of the anti-inflammatory/suppressive cytokine IL-10 bymonocytes and T cells, and induce the genera-tion of antigen-specific regulatory T cells. Theuse of ASCs for immunologic disorders, for
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example, in autoimmune-induced rheumatoidarthritis or inflammatory bowel disease, such asCrohn’s disease and ulcerous colitis, is currentlyunder investigation.
ASCs also protect from severe sepsis byreducing the infiltration of inflammatory cells invarious target organs and by downregulating theproduction of various inflammatory mediators.Colitic and septic mice were treated intraperi-toneally with human ASCs or murine ASCs, anddiverse clinical signs of disease were investi-gated as well as the levels of various inflam-matory cytokines and chemokines, T helper 1-type response and generation of regulatory Tcells in affected organs. Systemic infusion ofASCs significantly reduced the severity of colitisby eliminating weight loss, diarrhea, andinflammation, thereby increasing survival(Gonzalez-Rey et al. 2009).
9.6.15 Stem Cells in Graft-Versus-HostDisease
Twenty clinical trials of BMSCs for treatment ofGvHD have been reported (http://clinicaltrials.gov), of which 10 have already been completed.Yet, no clinical trials using ASCs for treatingsevere and acute GvHD are currently under way.Furthermore, the efficacy of ASCs is currentlybeing studied in GvHD; however, their effect onalloreactivity in solid organ transplant patients isunknown.
9.6.16 Stem Cells in VeterinaryMedicine
Stem cells play an important role in veterinarymedicine in different ways. Currently, severalstem cell therapies for animal patients are beingdeveloped and some, like the treatment ofequine tendinopathies with MSCs, have alreadysuccessfully entered the market. However, clin-ical applications of ES cells are not possible yetdue to their in vivo teratogenic degeneration.MSCs from either extra embryonic or adult
tissues are in the focus of attention in veterinarymedicine and research.
9.7 Advantages of Stem CellTherapy
Stem cell therapy is very promising and hasseveral advantages: (1) it is apparently not toxic;(2) it may be administrated once without theneed for repetition, (3) the progeny of trans-planted cells assume phenotypes in the targettissue, (4) the therapy may be applied withoutthe precise knowledge of mechanisms that con-trol cell homing, their proliferation and differ-entiation, and (5) the incurable diseases can alsobe treated by this therapy.
9.8 Disadvantages
Although the BMSCs continue to be a viableoption for a stem cell population for cell therapyapplications, there are drawbacks to utilizing thesource. A bone marrow harvest is a painfulprocedure with possible donor site morbidity asa result. Secondly, although MSCs grow wellunder standard tissue culture conditions, ex vivoexpansion is necessary due to relatively lownumbers of MSCs present in the harvestedmarrow.
There are several drawbacks which areassociated with iPS. These are based on the useof retroviruses and lentiviruses that integrateinto the DNA. In fact, there is correlationbetween the number of insertions of the transg-enes and the quality of the reprogramming(Okita et al. 2007). Viral integration on its owneither cause mutations or in the case of Klf4 andc-Myc, which are classified as oncogenes, thevectors become reactivated and cause tumors(Ton-That et al. 1997). Moreover, Oct-4 cancause dysplasia when overexpressed in adulttissues (Hochedlinger et al. 2005). Theoretically,use of nonintegrating viruses would be apotential solution; one clear example is adeno-viruses, while for lentiviruses DNA integration
176 S. Kulshreshtha and P. Bhatnagar
can be drastically reduced or eliminated bysimple mutation of the integrase viral gene. Inboth cases, the viruses will become progres-sively diluted with cell divisions and disappear.
9.9 Conclusion
Patients who are diagnosed with difficult to treatnervous system disorders, malfunctioning ordamage of any organ suffer tremulously, andoften desperately look for help and hope. In thisnew age of medical science, stem cell therapycan provide a ray of hope to these patients. Stemcells serve as sort of repair system. They cantheoretically divide without limit to replenishother cells for as long as the person or animal isstill alive. When a stem cell divides, each‘‘daughter’’ cell has the potential to eitherremain a stem cell or become another type ofcell with a more specialized function, such as amuscle cell, a red blood cell, or a brain cell.Some current therapies, such as bone marrowtransplantation, already make use of stem cellsand their potential for regeneration of damagedtissues. Other therapies are under investigationthat involves transplanting stem cells into adamaged body part and directing them to growand differentiate into healthy tissue. But, as it iswith any new advancement, people, who are nottoo familiar with it, try to use it for their owngain. Some of the treatment strategies are underclinical trials and their successful completionwill bring the technology to the field to treatvarious diseases and give new life to patients.Diseases and conditions, which were not possi-ble to treat before are now treatable and to someextent even curable by stem cell therapy.
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178 S. Kulshreshtha and P. Bhatnagar
10Biosensors
Mayank and Rachana Arya
Abstract
A biosensor is an analytical tool used for qualitative and quantitativedetection of analyte. It incorporates a biological recognition element suchas enzyme, microbial cell, receptor, DNA, antibody, antigen, etc., whichinteracts with the analyte to produce primary response. This initialresponse is identified and converted into a suitable electronic form withthe help of a suitable transducer device. Selection of transducer is basedon the type of signal obtained from the interaction of biologicalrecognition element and analyte. The commonly used devices areelectronic, optical, mechanical, and thermal transducer. To insureaccurate response, suitable immobilization techniques are required forbiological recognition elements, which provide proper interaction oftransducer with the signal obtained from primary sensing. Adsorption,cross-linking, entrapment, and covalent binding are some commonlyapplied techniques for immobilization. The electronic signal obtainedfrom transducer may easily be modified, amplified, displayed, andrecorded by suitable electronic devices. The use of biological componentfor sensing makes the biosensor very specific, fast, and reliable, thusbiosensors are nowadays utilized in various industries for monitoringsuch as food quality control, medical research, clinical diagnosis,environmental monitoring, agriculture, bioprocess monitoring and con-trol, and pharmaceutical industry, etc.
10.1 Introduction
In 1956, Leland C. Clark Jr., who is known as thefather of biosensors, published his definitivepaper on the oxygen electrode (Clark 1956).Based on this experience and addressing hisdesire to expand the range of analytes that couldbe measured in the body, he made a land markaddress in 1962 at New York Academy of
Mayank (&)Biochemical Engineering Department, BT KumaonInstitute of Technology, Dwarahat, Uttarakhand263653, Indiae-mail: [email protected]
R. AryaElectronics and Communication EngineeringDepartment, BT Kumaon Institute of Technology,Dwarahat, Uttarakhand 263653, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_10, � Springer India 2014
179
Science Symposium in which he described ‘‘howto make electrochemical sensors (pH, polaro-graphic, potentiometric, or conductimetric) moreintelligent’’ by adding ‘‘enzyme transducers asmembrane enclosed sandwiches’’. The conceptwas illustrated by an experiment in which glu-cose oxidase was entrapped at a Clark oxygenelectrode using dialysis membrane. The decreasein measured oxygen was proportional to glucoseconcentration (Clark and Lyons 1962). Clark’sideas became commercial reality in 1975, withthe successful launch of the Yellow SpringsInstrument Company (Ohio) glucose analyzer,based on the amperometric detection of hydrogenperoxide. This was the first of many biosensor-based laboratory analyzers to be built by com-panies around the world.
The term biosensor has been applied todevices either used to monitor living systems, orincorporating biotic elements. The consensus,however, is that the term should be reserved foruse in the context of a sensor incorporating abiological element such as an enzyme, antibody,nucleic acid, microorganism, or cell.
A typical biosensor consists of a biologicalcomponent (primary sensing element) and anelectronic device (transducer) that converts thebiological signal into a measurable output. Thebiological part of the sensor reacts with a par-ticular substance of interests (i.e., the analyte) toproduce a physical or biochemical change that isdetected and converted to an electrical signal bythe transducer. The amplifier increases theintensity of the signal so that it can be readilymeasured. These components are usually housedwithin a single portable unit that can be placed atfixed strategic locations. The biosensors displaycan be tailored to meet the needs of the appli-cation and can range from a simple output suchas switching on (or off), a light-emitting diode toa quantitative result displayed in graphical for-mat. The general schematic diagram of a bio-sensor is given in Fig. 10.1.
Biosensor consists of a biological entity suchas an enzyme, antibody or nucleic acid thatinteracts with an analyte and produces a signalthat is measured electronically. Each biosensor,therefore, has a biological component that acts
as the sensor and an electronic component totransduce and detect the signal. A variety ofsubstances including nucleic acids, proteins(particularly antibodies and enzymes), lectins(plant proteins that bind sugar moieties) andcomplex materials (organelles, tissue slices,microorganisms) can be used as the biologicalcomponents. In each case it is the specificity ofthe biological components for an analyte (orgroup of related analytes) that makes the bio-molecules attractive as sensing component. Forexample, a single strand of DNA can be used asa biomolecular sensor that will hybridize only toits complementary strands under appropriateconditions. The signal, which can be electrical,optical, or thermal, is converted by means of asuitable transducer into a measurable electricalparameter such as current or voltage. Biosensorprobes are attaining increasing sophisticationbecause of the fusion of two technologies:microelectronics and biotechnology. Biosensorsprovide a useful means for measuring a widespectrum of analytes (e.g., gases, ions, andorganic compounds, or even bacteria) and aresuitable for studies of complex microbialenvironments.
10.2 Overview of BiosensorTechnology
The important components of biosensor tech-nology and their interaction in the form of flowchart are represented in Fig. 10.2.
10.3 Biological RecognitionElements
The bioactive component could be an enzyme, anantibody, a cell (bacterial and fungal), a tissueslice, a receptor, nucleic acid, or an organelle. Itis used to recognize and interact specifically withthe analyte. Generally, there are two categoriesof biosensors, catalytic and affinity:1. Catalytic biosensors: In this type of biosen-
sors, biocatalysts such as enzyme, microbio-logical cells, animal and plant cells,
180 Mayank and R. Arya
organelles and tissues are used to recognize,bind, and chemically convert a moleculeidentified by the transducer. These biorec-ognition elements can be used to analyzesubstrate, inhibitor, activator, co-factor, andenzyme activity.
2. Affinity biosensors: In this category the bio-logical element (receptor) binds specifically tothe analyte leading to a complex. The trans-ducer reveals the complex and generates anelectronic signal. Receptor molecule such asantibodies, nucleic acids, lectins, and hormonereceptors are used to bind molecules irre-versibly and non-catalytically (Table 10.1).
10.3.1 Enzymes
Enzymes were the first biological componentsused in biosensors (Clark 1956) and remain byfar the most commonly employed elements.
Enzymes are the large protein molecules that actas biological catalysts, i.e., they speed upchemical reactions at moderate temperatureusually acting on a unique substrate or group ofsubstrates. It is this remarkable specificity thatmakes enzymatic methods attractive in sensingdevices. Enzymes are normally the preferredchoice in catalytic-type biosensor. However, inmany cases the use of enzymes is currentlyimpractical or impossible. The enzyme may beunstable, may require soluble cofactors, may bedifficult to purify, may not involve a readilydetectable species, or the analyte may not besubstrate for a specific enzyme.
10.3.2 Antibodies
In principle, an antibody-based assay can quantifyanalyte on the basis of binding reactions betweenantibody and antigen. It is done from a total of the
Electronic Signal
Impurities
Analyte
Semipermeable membrane: selective for analyte
Interaction of analyte with recognition element
Immobilized biological recognition elements
Product of recognition event
Passage of product to generate electronic signal
Semipermeable membrane: selective for product
Transducer
Amplifier
Display
Recorder
Samplesolution
Fig. 10.1 Schematicdiagram of biosensor
10 Biosensors 181
BIOCOMPONENTS
Enzyme, Microbial cells, Receptors, DNA, Antibodies, Antigens etc.
IMMOBILIZATION TECHNIQUES
AdsorptionCross linkingEntrapment
Covalent binding
TRANSDUCER
Electrochemical (hydrogen peroxide, oxygen, electron transfer)
Optical (absorbance, fluorescence, chemiluminescence)Thermal (temperature change, heat release)
Piezoelectric crystal (mass change, frequency)
APPLICATIONS
Food quality controlMedical researchClinical diagnosis
Environmental monitoringAgriculture
Bioprocess monitoring and controlPharmaceutical industry etc.
Biochemical signal
Electrical signal
AMPLIFIER, DISPLAY UNIT AND RECORDER
Fig. 10.2 An overview ofbiosensor technology
Table 10.1 Affinity biosensor
Device name Bio-recognition element Analyte
Immunoassay Antibody Antigen
– Receptor Hormone
– Enzyme Inhibitor
– Membrane transport protein Substrate analogue
Ion selective electrode Ionophore Ions
– Lectin Saccharide glycoprotein
– Apoenzyme Prosthetic group
182 Mayank and R. Arya
antibody-antigen complex after 20 min assayoperation. To shorten the time for the assay, a newformat was developed. The binding reactionsbetween antibody and antigen can be monitoredas a time-dependent change of fluorescence sig-nal. The signal change was proportional as areaction ratio of antibody to the analyte. Thus, itwas possible to monitor the analyte concentrationin the sample from the signal. As a result of themonitoring, the analytical time was dramaticallyshortened to less than 2 min.
10.3.3 Receptors
An active biological receptor can be immobi-lized and stabilized in a polymeric film to con-struct receptor-based biosensor for determiningan analyte of interest in a sample. The receptor-based biosensors include a polymeric film hav-ing a biological receptor capable of binding ananalyte of interest immobilized therein accord-ing to the method of the present invention and anelectrical means for determining the presenceand quantity of the analyte. In particular, ace-tylcholine and opiate receptors have beenimmobilized in a polymeric film made by com-bining the receptor, a material capable of poly-merization (e.g., bovine serum albumin, gelatin)and a polymerizing agent (e.g., glutaraldehyde).
10.3.4 Fragments of Nucleic Acids
An automated optical biosensor system was repor-ted based on fluorescence excitation and detectionin the evanescent field of a quartz fiber used todetect 16-mer oligonucleotides in DNA hybridiza-tion assays (Abel et al. 1996). For all hybridizationexperiments a biotinylated capture probe wasimmobilized using aminosilanized fibers.
10.3.5 Microorganisms
Microorganisms have a number of advantages asbiological sensing materials in the fabrication ofbiosensors. They are present ubiquitously and
are able to metabolize a wide range of chemicalcompounds. Microbes have a great capacity toadapt to adverse conditions and to develop theability to degrade new molecules with time.They are also amenable for genetic modifica-tions through mutation or recombinant DNAtechnology and serve as an economical source ofintracellular enzymes. A microbial biosensorconsists of a transducer in conjunction withimmobilized viable or non-viable microbial cells(Riedel 1998). Non-viable cells obtained afterpermeabilization or whole cells containingperiplasmic enzymes have mostly been used asan economical substitute for enzymes. Viablecells make use of the respiratory and metabolicfunctions of the cell, the analyte to be monitoredbeing either a substrate or an inhibitor of theseprocesses.
Microorganism-based biosensors tend to useone of three primary mechanisms. For the firstmechanism, the pollutant is a respiratory sub-strate. Biosensors that detect biodegradableorganic compounds measured as biologicaloxygen demand (BOD) are the most widelyreported of the microorganism-based biosensorsusing this mechanism. Several of these devicesare commercially available from vendorsincluding: Nisshin Electric Co. Ltd., Tokyo;Autoteam, GmbH, Berlin; Prufgeratewrk, Med-ingen GmbH, Dresden; and Dr. Lange, GmbH,Berlin. The use of these devices has beenincorporated into industrial standard methods inJapan.
Another mechanism used for microorganism-based biosensors involves the inhibition of res-piration by the analyte of interest. Microbialrespiration and its inhibition by various envi-ronmental pollutants have been measured bothoptically and electrochemically. Inherentadvantages of these techniques apply primarilyto the use of microorganisms as compared toisolated enzymes. These biosensors are rela-tively inexpensive to construct and can operateover a wide range of pH and temperature.General limitations involve the long assay timesincluding the initial response and return tobaseline. These characteristics are primarilydetermined by the cellular diffusion
10 Biosensors 183
characteristics that can be modified by usinggenetically engineered microorganisms. Thebroad specificity of these biosensors to envi-ronmental toxins may be an advantage or dis-advantage depending on the intendedapplication. In this respect, these devices mightbe most applicable for general toxicity screeningor in situations where the toxic compounds arewell defined, or where there is a desire to mea-sure total toxicity through a common mode ofaction.
Biosensors have also been developed usinggenetically engineered microorganisms (GEMs)that recognize and report the presence of specificenvironmental pollutants. The microorganismsused in these biosensors are typically producedwith a constructed plasmid in which genes thatcode for luciferase or b-galactosidase are placedunder the control of a promoter that recognizesthe analyte of interest. Because the organism’sbiological recognition system is linked to thereporting system, the presence of the analyteresults in the synthesis of inducible enzymeswhich then catalyze reactions resulting in theproduction of detectable products. With respectto environmental applications, the primary dis-advantage for this type of biosensor is the lim-ited number of GEMs which have beenconstructed to respond to specific environmentalpollutants. Nevertheless, reported advancesinclude the development of GEMs involving avariety of bioremediation pathways and mecha-nisms. GEMs that could report both the meta-bolic condition of the relevant microorganismsas well as the rates of pollutant breakdown couldbe very useful (Burlage and Kuo 1994).
10.3.6 Lectin
Lectins are proteins which are highly specificfor their sugar moieties. They play a role inbiological recognition phenomena involvingcells and proteins. Specific mono- and oligo-saccharides bind to lectins and provide theirattachment to cell membranes. Microbial surfacebear many of the sugar residues capable of
interacting with lectins. The ability of lectins toreact with microbial glycoconjugates rendersthem to be employed as probes and sorbents forwhole cells, mutants, and numerous cellularconstituents and metabolites. Lectins are attrac-tive reagents for the clinical diagnostic labora-tory because of their diverse specificity,commercial availability, a wide range ofmolecular weights, and their stability in standardbuffers. A lectin-based biosensor is reported forelectrochemical assay of glycan expression onliving cancer cells which may facilitate themedical diagnosis and treatment in early processof cancer (Zhang et al. 2010).
10.3.7 Membrane Transport Protein
A membrane transport protein is a membraneprotein involved in the movement of ions,small molecules, or macromolecules, such asanother protein across a biological membrane.Transport proteins are integral membrane pro-teins; that is they exist within and span themembrane across which they transport sub-stances. The proteins may assist in the move-ment of substances by facilitated diffusionor active transport. These mechanisms of actionare known as carrier-mediated transport. Aprototype lactose sensor is reported which con-sists of a quartz slide covered by a lipid mem-brane containing the protein lactose permeasefrom Escherichia coli. This protein is a lactose/H+ co-transporter, hence lactose in the externalmedium initiates lactose/H+ co-transport acrossthe lipid membrane. This leads to a rise in protonconcentration in the small volume between thelipid membrane and the quartz surface whichcan be detected by a pH-sensitive fluorescencedye (Kiefer et al. 1991).
10.4 Transducers
The transducing element of a biosensor is usedto convert the biological recognition step into anelectrical signal that can be coupled to a
184 Mayank and R. Arya
microprocessor for control and display. Trans-ducers are mainly divided into four major cate-gories as summarized in Table 10.2.
10.4.1 Electrochemical Transducers
The most common transducer for the construc-tion of a biosensor is electrochemical transducer.The electrochemical device uses a chemicalchange to measure the input parameter, whereasthe output is a varying electrical signal propor-tional to the measured chemical change. On thebasis of output electrical signal the electro-chemical transducers may be classified as:
10.4.1.1 Amperometric BiosensorsThese biosensors are based upon the resultingsteady state limiting current between two elec-trodes under constant voltage. Most of the glu-cose biosensors are based upon this principle. Itcan be either directly or indirectly mediatedusing suitable redox mediator like ferrocene dye.The electrode potential E will control the con-centration of the two redox forms according toNernst equation:
E ¼ E0 þ RT=nF ln ox½ �= red½ �f g
E0 is electrode potential at standardconditions
R is universal gas constantT is absolute temperatureF is Faraday constantn is electrons transferred in the
reaction[ox]/[red] is ratio of oxidized to reduced
molecules
10.4.1.2 Potentiometric BiosensorsThese biosensors are based upon non-faradicelectrodes process with no net current flow.They are based on the rise in electrode potentialdue to the accumulation of the ionic speciesproduced from the particular biochemical reac-tions and is measured with a reference electrode.This electrode potential is proportional to ana-lyte concentration in log scale and is also gov-erned by Nernst equation. These sensors haveaccordingly glass electrode, ion selective elec-trode, or field effect transistor (FET) as thetransducer. Many biosensors for b-lactam anti-biotic are based upon this principle.
10.4.1.3 Impedance BiosensorsImpedance biosensors may be used to measurethe electrical impedance of an interface in ACsteady state with constant DC bias conditions.Mostly this is accomplished by imposing a smallsinusoidal voltage at a particular frequency and
Table 10.2 Summary of transducer devices
Type Device Parameter measured
Electronic Amperometric Change in current
Potentiometric Change in potential
Impedance Change in impedance
Thermal Thermistor Change in temperature
Mechanical Piezo-electric Change in mass vibrations
Acoustical Acoustical Change in amplitude, phase, or frequency ofacoustic wave
Optical (Colorimetric andPhotometric)
Photo-conductive Change in intensity, colour or emission
Photo-emissive Fluorescence or luminescence activation,quenching, and polarizationPhoto-diodes
Photo-transistors
Fluorescence orChemiluminiscence
10 Biosensors 185
measuring the resulting current; the process canbe repeated at different frequencies. The cur-rent–voltage ratio gives the impedance and thistechnique is known as electrochemical imped-ance spectroscopy (EIS). The impedance of theelectrode-solution interface changes when thetarget analyte is captured by the probe. EIS canbe utilized over a wide frequency range or at asingle frequency to study a variety of electro-chemical phenomena. Impedance measurementdoes not require special reagents or labels foroperation.
10.4.2 Optical Transducers
Optical sensors, originally developed for oxygen,carbon dioxide, and pH, have been extended tothe construction of fluorescent and luminescentopto-electrodes. The selective biocomponent isimmobilized at one end of optical fiber, with boththe excitation and detection components locatedat the other end. NAD(P)+/NAD(P)H- dependentdehydrogenases are obvious candidates for thesebiosensors, because NAD(P)H is known toabsorb light strongly at 340 nm and fluorescenceat 460 nm. Several NAD(P)+/NAD(P)H-depen-dent dehydrogenase enzymes exist for a numberof food and beverage-related compounds, but therequirement for expensive and unstable cofactorsis the major drawback of this technique. Hydro-gen peroxide fiber-optic sensor based on absor-bance or chemiluminescence has also beendeveloped for various analytes using oxidases.Optical transducers can also take advantages ofaffinity interactions between antibodies andantigens to screen for microbial contaminants(Mehrvar et al. 2000).
10.4.3 Thermal Transducers
This device is regarded as a small microcalo-rimeter with immobilized biocomponents,packed in a small column in proximity to the heatsensing transducer, commonly a thermistor. Mostenzyme or microorganism catalyzed reactions
are accompanied by considerable heat evolution(20–100 kJ/mol), hence this device has a broaderapplicability than other transducers. The com-mercial enzyme thermistor claims to detecttemperature change in the range 0.0001–0.05 �C.The sensitivity of this technique is equivalent tothe substrate concentration of 10-5 M. Thermaltransducers have also been applied for thedetection of antibody-antigen interactions. Thistechnique is known as the thermometric enzymelinked immunosorbent assay, however its use issophisticated and expensive.
10.4.4 Piezoelectric Transducer(Quartz Microbalance)
A quartz crystal can oscillate when partially orcompletely immersed in a liquid, and if thephysiochemical properties of the liquid (such asviscosity, density, and conductivity) are defined,a change in the weight of the quartz crystal canbe detected by measuring its vibration frequency.Biologically active materials such as antibodies,enzymes, and antigens have been used as activecoatings. For the past two decades, intensiveresearch has been directed to developing piezo-electric sensors for a variety of applications.However, non-specific binding and poor sensi-tivity are the two major drawbacks of piezo-electric transduction (Sochaczewski et al. 1990).
10.5 Immobilization Techniques
An efficient and effective biosensor requiresintimate contact between its component parts,normally achieved by restricting the biomole-cule, cell, or organelles to a thin layer on or nearthe transducer surface. The processes employedin achieving this restriction are collectivelyknown as immobilization techniques, definedaccording to the recommendation of standardi-zation of nomenclature in enzyme technology as:1. Entrapment with a membrane, gel, or
microcapsule.2. Physical or chemical adsorption.
186 Mayank and R. Arya
3. Cross-linking between molecules.4. Covalent bonding to soluble or insoluble
supports.The localization of biological components at
the transducer confers several advantages:1. Enables recovery of the biocomponent with-
out dilution or contamination.2. Permits continuous or repeated use of the
sensor.3. Maximizes response by concentrating the
biocomponent.4. Often results in improvement of enzyme
stability due to increased rigidity (whichmaximizes unfolding), a favorable microen-vironment, or diffusional effects (Klibnov1983).The original concept of biosensor used
enzyme entrapment at an electrode with dialysismembrane. This simple method has generallybeen superseded by other techniques but is stillemployed with enzymes and microbial cells.Enzyme entrapment within a polyacrylamide gelmatrix was successfully employed by Updikeand Hicks (1967). This method has been widelyadopted. Despite its popularity as an enzymeimmobilization technique, gel entrapment isbetter suited for cells and organelles sinceenzyme molecules tend to leak from the matrix.Other polymers used for the occlusion of cellsand/or enzymes include alginate, carrageenan,agar, cellulose acetate, polyvinyl alcohol (PVA),polyvinyl chloride, polycarbonate, and polypyr-role (Brooks et al. 1991). The procedure withtheir inherent advantages and disadvantages arepresented in Table 10.3.
Adsorption is the oldest known form ofimmobilization, dating back to 1916 when Nel-son and Griffin first immobilized invertase onalumina and charcoal. Adsorption in aqueousphase is a complex process involving hydro-phobic interactions, physical adsorption, andchemisorption. Physical adsorption is a result ofVan der Waals forces which are made up ofLondon dispersion forces and electrostaticinteraction. In contrast to physical adsorptionwhere there is no net transfer of electrons fromthe interacting species, in chemisorption there is
a sharing or transfer of electrons to form achemical bond, for example via pi–pi bonding.Biosensor applications of adsorption techniquesare limited by their reversibility, since thedegree of attachment is often insufficient tomaintain a constant coverage during repeated orcontinuous uses (Riedel 1998).
Copolymerization of enzymes can beaccomplished by cross-linking with a lowmolecular weight bi-functional reagent, resultingin a soluble film which adheres to, or is retainedat, a transducer. In biosensor applications, glu-taraldehyde, which couples to lysine residues, isby far the most popular of the numerous bi-functional reagents that have been developed forcross-linking of proteins. A lysine rich protein,such as albumin or collagen, is often added toglutaraldehyde-enzyme preparations to increasethe number of cross-linking sites and to providephysical strength. Bi-functional reagents areavailable for modification of other proteinfunctional groups, including carboxyl, sulfhy-dryl, phenol, and imidazole groups (Deshpandeet al. 1986; Svitel et al. 1998).
Some remarkably stable and durable immo-bilized enzyme preparations have been achievedby the covalent attachment of biological mole-cules to a solid surface, such as a membrane,bead or tube, which is positioned at or near thetransducer surface. Support materials can bebroadly classified into inorganics (glasses, tita-nium dioxide), polysaccharides (starch, agarose,cellulose), and other polymers (nylon, polyami-nostyrene, and polymethacrylate). A bifunc-tional reagent is covalently bonded to thesupport and the enzyme subsequently reactedwith the activated surface. The permanence ofcovalent attachment is a great advantage in thefabrication of enzyme sensors. However, suchimmobilization may be deleterious as thereagents used can cause an initial loss of activitydue to inadvertent modification of amino acidresidues at or near the site. Alternatively, gly-coproteins such as glucose oxidase can be cross-linked or bound to a surface via activated car-bohydrate residues with the advantage that theactive site remains unchanged (Scouten 1987).
10 Biosensors 187
Matrix material, nature of bonding, the cost ofimmobilization is compared for various tech-niques in Table 10.4.
10.6 Characteristics of Biosensors
10.6.1 Response Time
When, there is a change in input parameter,biosensors do not change output state immedi-ately. Rather, it will change to the new state overa period of time, called the response time. Theresponse time can be defined as the timerequired for a sensor output to change from its
previous state to a final-settled value within atolerance band of the correct new value. Thisconcept is somewhat different from the notion ofthe time constant of the system.
10.6.2 Precision and Accuracy
Accuracy is a measurement of how close thesensor comes indicating the actual value of themeasured variable. Accuracy is always given interms of inaccuracy such as ±1 %. Precision is ameasure of the consistency of a sensor in mea-suring the same value under the same operatingconditions over a period of time.
Table 10.3 Immobilization procedure for biosensor construction
Methods Advantages Disadvantages
Adsorption on insolublematerials (Van der waal’sforces, hydrophobic forces, orionic bonding)
Simple, mild conditions, and mildtoward proteins
This technique is highly sensitivetoward pH, solvent, and temperature
Cross-linking (usingmultifunctional groups)
Simple technique, strong chemicalbinding of the biomolecules,physically adsorbed proteins(covalently bound onto a support) arestabilized with this technique
Relatively low enzyme activity sorequires more enzymes, reactioncontrol is difficult, the protein layer isgelatinous, provides less rigidity
Entrapment (gel or semipermeable membrane)
Mild procedure and commontechnique for any enzyme
Mass transfer resistance is high,leakage of biocatalyst, biocatalystmay denature as a result of reaction
Covalent binding (membrane orinsoluble supports)
Stable enzyme-support complex,leakage of the biomolecule is rare,ideal for mass production andcommercialization
Intricate and time consumingprocedure, activity may decrease dueto reaction with groups essential forthe biological activity (can beovercome by immobilization in thepresence of the substrate or inhibitorof enzyme)
Table 10.4 Summary of immobilization techniques
Adsorption Entrapment Covalent coupling Cross-linking
Matrixmaterial
Ion exchange resins,active charcoal, silicagel, clay, aluminumoxide, porous glass
Alginate, carageenan,collagen,polyacrylamide,gelatine, silicon rubber,polyurethanes
Agarose, cellulose,Polyvinyl Chloride,ion exchange resins,porous glass
Cross linking agents:glutaraldehyde, bis-isocyanate, bis-diazobenzidine
Natureofbonding
Reversible; changes inpH, ionic strength maydetach the enzyme
Physical entrapment Chemical bonding Entrapment;functionally inertproteins are oftenused together (BSA,gelatin)
Cost Inexpensive Inexpensive Expensive Inexpensive
188 Mayank and R. Arya
10.6.3 Reproducibility
Reproducibility has the same definition forelectrochemical biosensors, as any analyticaldevice. It is the measure of the scatter or of thedrift in a series of observation or result per-formed over a period of time. It is a generallydetermined for analyte concentrations with theusable range.
10.6.4 Lifetime
Although some biosensors have been reportedusable under laboratory condition for periods ofmore than 1 year, their practical life time whenincorporated into industrial processes or to bio-logical tissue, such as glucose biosensorsimplanted in vivo, is either unknown or limitedto days or weeks. While it is relatively easy todetermine the laboratory bench stability of bio-sensors, both during storage and operation in thepresence of analyte, procedure for assigningtheir behavior during several days of introduc-tion into industrial reactors is much complex anddifficult to handle. In both cases one shouldspecify whether lifetime is a storage (shelf) oroperational (use) one and what are the storageand operating conditions. Finally the mode ofassessment of lifetime should be specified, i.e.,by reference to initial sensitivity, within thelinear range of the calibration curve, to decreaseby 10 % (LT 10) or 50 % (LT 50). For thedetermination of the storage life time, it is sug-gested to compare sensitivities of different bio-sensors, issued from the same production batchof homogenous patterns, after different storageduration time under identical conditions. Term‘lifetime’ is still the topic of ongoing discussion.
10.6.5 Stability
The operational stability of a biosensor responsemay vary considerably depending on the sensorgeometry, method of preparation, as well as onthe applied receptor and transducer.
Furthermore, it is strongly dependent upon theresponse rate-limiting factor, i.e., substrateexternal/inner diffusion or biological recognitionreaction. Finally, it may vary significantlydepending upon the operational conditions suchas continuous/discontinuous contact with sub-strate containing solution, substrate concentra-tion, temperature, pH, presence of organicsolvents, and the sample matrix.
Within the biosensor community the term‘‘biosensor stability’’ is controversial. However,it has been agreed that a general definition wouldbe useful and beneficial. Without a generalagreement it would be almost impossible todirectly compare ‘stability data’ obtained fromdifferent experimental setups at differentresearch centers.
1. Operational stability depends upon fol-lowing factors:(a) Response rate-limiting factor: mass
transfer or catalytic limiting(b) Continuous versus discontinuous con-
tact with substrate containing solution(c) Substrate concentration(d) Temperature(e) Buffer(f) Organic solvents(g) Sample matrix
2. Storage stability depends on followingfactors:(a) Dry or wet storage(b) Air or nitrogen atmosphere(c) Temperature(d) Buffer(e) Additives.
10.6.6 Drift Rate
Alternatively, biosensor stability measure may bequantified as a drift rate, when the sensitivityevolution is monitored during storage or opera-tional conditions. Drift rate is defined as the slowchange in output signal, independent of measuredproperty. It is especially useful for biosensorwhere sensitivity evolution is either very slow orstudied during rather short periods of time.
10 Biosensors 189
10.6.7 Specificity
Like other bioanalytical methods (such asimmuno-assays and enzyme-assays), biosensorsuse a biologically derived compound as thesensing elements. The advantage of biologicalsensing elements is their remarkable ability todistinguish between the analyte of interest andsimilar substances. With biosensors, it is possi-ble to measure specific analytes with greataccuracy.
10.6.8 Speed
One characteristic of biosensors that distinguishthem from other bioanalytical method is that theanalyte tracers or catalytic products can bedirectly and instantaneously measured. There isno need to wait for results from lengthy proce-dures carried out in centralized laboratories.
10.6.9 Simplicity
The uniqueness of a biosensor is that thereceptor and transducer are integrated into onesingle sensor. This combination enables themeasurement of target analytes without usingreagents. For example, glucose concentration ina blood sample can be measured directly by abiosensor (which is made specifically for glu-cose measurement) by simply dipping the sensorin the sample. This is in contrast to the con-ventional assay in which many steps are usedand each step may require a reagent to treat thesample.
10.6.10 Online Monitoring
Another advantage that biosensors have overbioanalytical assay is that they can regenerateand reuse the immobilized biological recogni-tion component. For enzyme-based biosensors,an immobilized enzyme can be used for repeatedassays: this feature allows these devices to be
used for continuous or multiple assays. Bycontrast, immunoassay including enzyme-linkedimmunosorbent assay (ELISA), are typicallybased on irreversible binding and are thus usedonly once and then discarded.
10.7 Applications of Biosensors
Biosensors are being developed for differentapplications including environmental and bio-process monitoring and control, quality controlin food industry, agriculture, military, and par-ticularly, medical uses. In fact, most of thecommercially available biosensor systems areapplied in the clinical and pharmaceutical mar-kets. A few applications of biosensors are sum-marized in Table 10.5.
10.7.1 Food and PharmaceuticalQuality Control
Food microbiologists are constantly seekingrapid and reliable automated systems for thedetection of biological activity. Biosensors pro-vide sensitive, miniaturized systems that can beused to detect unwanted microbial activity or thepresence of a biologically active compound,such as glucose or a pesticide. The use of im-munodiagnostics and enzyme biosensors hasreduced the time for detection of pathogens suchas Salmonella and has provided detection ofbiological compounds such as cholesterol orchymotrypsin. Improving the preservation orincreasing the yield of agricultural products is acommon aim for the producers, normally real-ized by using herbicides, insecticides, antibiot-ics, or hormones. The uses of these xenobioticsare a potential risk for consumers. The safetyand quality of the food is a major concern thesedays. In novel foods such as functional foods,ingredients or constituents are present to tacklehealth and bad eating-habit-related issues. In allthese cases there is the need of monitoring anincreasing number of various analytes. Thecontinued research in biosensor technology will
190 Mayank and R. Arya
Table 10.5 Applications of microbial biosensors in food, fermentation, environmental, and allied fields
Analyte Microorganism Transducer/immobilization
Reference
BOD Pseudomonas putida Oxygen electrode(adsorption on porousnitro cellulosemembrane)
Chee et al.(1999)
BOD Trichosporum cutaneum Miniature oxygenelectrode (UV cross-linking resin (ENT-3400)
Yang et al.(1996)
BOD Activated sludge (mixed microbialconsortium)
Oxygen electrode/flowinjection system(entrapped in dialysismembrane)
Liu et al.(2000)
BOD Salt tolerant mycelial yeast Oxygen electrode (PVA) Tag et al.(2000)
Anionic surfactants[linear alkyl benzenesulfonates (LAS)]
LAS degrading bacteria isolated fromactivated sludge
Oxygen electrode,(reactor type sensor, Ca-alginate)
Nomura et al.(1994)
Phenolic compounds Pseudomonas putida Oxygen electrode(reactor with cellsadsorbed on PEI glass)
NandakumarandMattiasson(1999)
Nitrite Nitrobacter vulgaris Oxygen electrode(adsorption on Whatmanpaper)
Reshetilovet al. (2000)
Cyanide Saccharomyces cerevisiae Oxygen electrode (PVA) Ikebukuroet al. (1996)
Chlorophenols Rhodococcus sp.; Trichosporon beigelii Oxygen electrode (PVA) Riedel et al.(1993, 1995)
Organophosphate nerveagents (paraxon, methylparathion, diazinon)
Genetically engineered Escherichia coli(organophosphorous hydrolase)
Potentiometric(adsorption on electrodesurface)
Mulchandaniet al. (1998)
Alcohol Candida vini Oxygen electrode(porous acetyl cellulosefilter)
Mascini et al.(1989)
Glucose Aspergillus niger (glucose oxidase) Oxygen electrode(entrapment in dialysismembrane)
Katrlik et al.(1996)
Glucose, sucrose,lactose
Gluconobacter oxydans (D-glucosedehydrogenase), Saccharomycescerevisiae (invertase), Kluyveromycesmarxianus (b-galactosidase)
Oxygen electrode(gelatin)
Svitel et al.(1998)
Short chain fatty acidsin milk (butyric acid)
Arthrobacter nicotianae (acyl-CoAoxidase)
Oxygen electrode(Polyvinyl alcohol)
Ukeda et al.(1992a, b)
Phosphate Chlorella vulgaris Oxygen electrode(polycarbonatemembrane)
Matsunagaet al. (1984)
CO2 CO2 utilizing autotropic bacteria(Pseudomonas)
Oxygen electrode (boundon cellulose nitratemembrane)
Suzuki andKarube(1987)
Vitamin B-6 Saccharomyces uvarum Oxygen electrode(adsorption on cellulosenitrate membrane)
Endo et al.(1995)
(continued)
10 Biosensors 191
soon provide on-line quality control of foodproduction, which will not only reduce the costof food production but will also provide greatersafety and increased food quality.
The pharmaceutical industry is constantlylooking for new tools, techniques and practicesto increase the productivity of research anddevelopment and to improve the quality of theinnovative medicines they produce. Biosensorscomprise a very specific biological componentsuch as enzyme, antibody receptor etc., thusprovide an advantage of quantitative study of theinteraction between a drug compound and animmobilized biocomponent. Affinity biosensorsare suitable for high-throughput screening ofbioprocess produced antibodies and for candi-date drug screening. Enzyme- based biosensorscan be applied in the pharmaceutical industry formonitoring chemical parameters in the produc-tion processes.
10.7.2 Medical Research and ClinicalDiagnosis
The initial thrust for advancing biosensor tech-nology came from health care area, wherediagnostics and monitoring have key roles to
play in optimising health care. It is now gener-ally recognized that measurements of bloodgases, ions, and metabolites are often essentialand allow a better estimation of the metabolicstate of a patient. There is still a notable gapbetween laboratory biosensing and commer-cially viable medical or consumer diagnosticdevices. The biosensor community needs torefine its work to the wider population for tele-medicine or telehealthcare.
10.7.3 Environmental Monitoring
Another assay situation which may involve aconsiderable degree of the unknown is environ-mental monitoring. The primary measurementmedia here will be water or air, but the variety oftarget analytes is vast. At sites of potential pol-lution, such as factory effluent, it would bedesirable to install on-line real-time monitoringand alarm, targeted at specific analytes, but inmany cases random or discrete monitoring ofboth target species and general hazardous com-pounds would be sufficient. The possible ana-lytes include biological oxygen demand (BOD)which provides a good indication of pollution,atmospheric acidity, and river water pH,
Table 10.5 (continued)
Analyte Microorganism Transducer/immobilization
Reference
Vitamin B-12 Escherichia coli Oxygen electrode(trapped in porous acetylcellulose membrane)
Karube et al.(1987)
Peptides (aspartame) Bacillus subtilis Oxygen electrode (filterpaper strip and dialysismembrane)
Renneberget al. (1985)
Phenylalanine Proteus vulgaris (Phenylalaninedeaminase)
Amperometric oxygenelectrode (Ca-alginate)
Liu et al.(1996)
Pyruvate Streptococcus faecium (Pyruvatedehydrogenase complex)
CO2 gas sensingelectrode (directimmobilization on sensormembrane)
DiPaolantonioand Rechnitz(1983)
Tyrosine Aeromonas phenologenes (Tyrosine-phenol lyase)
NH3 gas sensingelectrode (directimmobilization on sensormembrane)
DiPaolantonioand Rechnitz(1982)
Enalapril maleate(angiotensin)
Bacillus subtilis Oxygen electrode Fleschin et al.(1998)
192 Mayank and R. Arya
detergent, herbicides, and fertilizers (organo-phosphates, nitrates, etc.). Biosensors can beexcellent analytical tools for monitoring pro-grams working to implement legislation.
10.7.4 Agriculture
Biosensor technology is a powerful technique,having the specificity and sensitivity of biolog-ical systems in small and low cost devices formeasurement. Though various biosensors aredeveloped in research laboratories, there are notmany reports of applications in agriculturalmonitoring. The need for fast, on-line, andaccurate sensing opens up opportunities forbiosensors in many different agricultural areassuch as in situ analysis of pollutants in crops andsoils, detection and identification of diseases incrops and livestock, monitoring animal fertilityand screening therapeutic drugs in veterinarytesting and the detection of various compoundsaffecting the fertility of soil.
10.7.5 Bioprocess Monitoringand Control
While real-time monitoring with feedback con-trol involving automated systems does exist,currently a few common variables are measuredon-line (e.g., pH, temperature, CO2, O2) whichare often indirectly related with the processunder control. Real-time monitoring of carbonsources, trace metals, dissolved gases, importantcomponents of media, and products in fermen-tation processes could lead to optimization of theprocedure giving increased yields at decreasedmaterials cost.
10.8 Conclusion
In this chapter we have discussed various types ofbiosensing elements, transducers, and immobili-zation techniques utilized to develop biosensors.The diversity of these biosensor componentsprovides opportunity to develop biosensors for a
variety of analytes and thus can be used in diverseapplications. The activity and specificity of bio-sensing elements make them suitable for analysisof complex solutions with short response time.Various biosensors are having the capability to beused for online analysis which enhances the useof biosensors in food and bioprocess industries tooptimize production, in medical field for screen-ing of the potential drug molecules and in envi-ronmental field to identify and quantify thepotential pollutants. The efforts are continuouslymade to develop new biosensors and commer-cialize them but still few biosensors are com-mercially successful and there is a vast scope ofresearch and development in this sector.
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11Monoclonal Antibody Productionand Applications
B. D. Lakhchaura
Abstract
Antiserum is a heterogeneous mixture of antibodies produced against anantigen. Antigens usually have multiple epitopes and separate antibodiesare produced against each by an individual clone of B cell. Monoclonalantibody produced by hybridoma technique is a single homogeneousantibody population which is specific for one epitope and exhibits nocross-reaction. The technique involves fusion of B cell with the myelomacell and cultivation of the fused clone for indefinite production of thedesired antibody. Myeloma cells are the tumor cells and impartimmortality to the clone while B cells contribute to the antibodyproduction ability. Selection of the fused cell is carried out inhypoxanthine-aminopterin-thymidine (HAT) containing medium so thatunfused myeloma cells are unable to grow by virtue of their HGPRTnegative character. Hybridomas once produced can be cryopreserved inliquid nitrogen for indefinite storage. It is possible to antibodies in culturemedium or as ascitic fluid in mice. Both procedures allow sizeableharvest of the antibody. Monoclonal antibodies have proven to bevaluable tools in immunodiagnostic, immunotherapy, and in biologicaland biochemical research. Monoclonal antibody-based immunodiagnos-tic kits are available for detection of pregnancy, for diagnosing numerouspathogenic microorganisms, measuring level of drug in blood or urine,matching histocompatibility antigens, and detecting antigens shed byvarious tumors. A sizable number of monoclonal antibodies are availableas therapeutic agents.
11.1 Introduction
Antibodies play an important role in bodydefence against infectious diseases. Antibodiesare glycoprotein and are produced by B lym-phocytes with the help of helper T lymphocytesand antigen presenting cells. An antigen, when
B. D. Lakhchaura (&)G. B. Pant University of Agricultureand Technology, Pantnagar, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_11, � Springer India 2014
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administered in an appropriate vertebrate host,results in eliciting an immune response. Basi-cally there are two types of immune responses,humoral and cell mediated. In humoral response,B lymphocytes are activated by antigen pre-senting cells and helper T lymphocytes. On theother hand, cell-mediated immune responserequires activation of cytotoxic T lymphocytes.An antigen molecule possesses a large numberof antigenic determinants which are called epi-topes. Each antibody is specific for a particularepitope. Thus the antisera raised against a givenantigen are a mixture of antibodies, each specificfor an epitope. Such antisrea are known aspolyclonal antisera. As all antibody moleculesare similar in their physical and chemical prop-erties, it is impossible to separate epitope spe-cific antibodies from each other. Antibodiesproduced from a single clone of B lymphocyteare called as monoclonal antibody (MAb).
The theory of monoclonal antibody productionis based on the clonal selection hypothesis of F.Macfarlane Burnet (1959). Each mammalian Blymphocyte has the potential to make a mono-specific antibody. The constant region of theantibody chain may alter during the differentia-tion of the lymphocyte clone but the variableregion retains this singular specificity. The firstreport of Hybridoma production was in fact in1970 (Sinkovics et al. 1970). Kohler and Milsteinin 1975 developed a technique that made it
possible to raise epitope specific antibodies; thistechnique was christened as Hybridoma Tech-nology and the antibodies produced by thismethod are called Monoclonal antibodies.Table 11.1 gives a comparison between conven-tional serum and monoclonal antibodies.
11.2 Production of MonoclonalAntibodies
Antibodies are secreted by plasma cells whichare programmed in lymph glands for producingantibodies against a particular epitope. Theplasma cells can be cultured and made to secreteantibodies but they have a very short life spanand die soon. The technique developed byKohler and Milstein (1975) immortalizes theplasma cells which can thus produce monoclonalantibodies indefinitely. For this purpose theyfused the plasma cells with cancerous cellscalled myeloma cells. Myeloma cells can beobtained from animals suffering from myelomatumors or tumors can be induced in experimentalanimals by injecting mineral oil. They used aninbred strain of mice known as Balb/c mice forthis purpose. A schematic illustration formonoclonal antibody production is given inFig. 11.1. All the steps are also detailedsubsequently.
Table 11.1 Comparison between conventional serum and monoclonal antibodies
S. N. Property Conventional antiserum Monoclonal antibody
1. Determinant Several Single
2. Specificity Variable with animal and bleed Standard
Partial cross-reactions withcommon determinants
Unexpected cross-reactions may occur
Seldom too specific May be too specific for requirements
3. Affinity Variable with bleed May be selected during cloning
4. Yield of usefulantibody
Up to 1 mg/ml Up to 100 lg/ml in tissue culture, Up to20 mg/ml in ascitic fluid
5. ContaminatingImmunoglobulin
Up to 100 % None in culture, 10 % in ascetic fluid
6. Purity of antigen Either pure antigen or serumabsorption
Some degree of antigen purificationdesirable but not essential.
7. Approx. Minimumcost
Usually below £100 Capital cost £10,000, Runningcost £10,000 p.a.
196 B. D. Lakhchaura
11.3 Monoclonal Antibody AssayRequirements
Assay is the most critical factor in production ofgood hybridomas and is very sensitive. Under-standing of the theoretical background isessential for the production of a large number ofhybridomas secreting antibody of the requiredcharacteristics.a. Rate of association
The initial rate of association of an antibodywith an antigen is described by the equation:
Rate of formation of product¼ K1½antibody�½antigen�
The number of epitopes on the antigen ismuch reduced and may be one in case of smallprotein. Thus the effective concentration of theantigen is quite low. The antibody will still bebivalent or decavalent for IgM but its concen-tration may be very low. The association rate is
usually the parameter which determines the finalequilibrium constant and the concentration ofantigen in most assays remains constant.b. Rate of dissociation
The rate of dissociation of an antibody-anti-gen complex is represented by the followingequation:
Initial rate of dissociation¼ K2ð½antibody� antigen�Þ
The dissociation rate is nearly always verymuch lower than the association rate. With mostantibody-antigen reactions, it is the dissociationrate rather than the association rate that deter-mines the affinity as the dissociation rate canvary over 8–9 orders of magnitude. The disso-ciation rate is not only the most variable amongdifferent antibodies under a defined set of con-ditions but also the most variable in a singleantibody with respect to environmental condi-tions such as pH, temperature, etc.
Fig. 11.1 Schematicrepresentation ofmonoclonal antibodyproduction
11 Monoclonal Antibody Production and Applications 197
c. Equilibrium concentration of reactantsThe equilibrium constant for an antibody-
antigen reaction is the ratio of the forward to thebackward rates i.e.,
Keq ¼ K1=K2 or
Keq ¼ ðAb� Ag)=ðAbÞðAgÞ
The concentration of the reactants and theassay conditions may influence the possibility ofdetection of a suitable hybridoma. With poly-clonal sera it can be assumed that an optimalequilibrium condition will be achieved byincubation for one or two hours at room tem-perature, biological pH, and ionic strength, etc.but for monoclonal antibodies several of theparameters will have to be varied and screeningprocedures will have to be extended if antibodiesto more interesting or relevant epitopes are to bedetected.d. Effect of multivalence
All antibodies are at least divalent and IgAand IgM antibodies may have several idiotypeson the same molecule. Multivalent antibodiesare helpful to screening procedures since theeffective dissociation rate can be reduced ifenough antigens are present. Antigens havingmultiple epitopes, mostly found in bacterialsystems with symmetrical cell wall structures,can affect the reaction in many ways.e. Specificity and affinity
Monoclonal antibodies against polymorphicantigens could exhibit extreme specificity in oneassay and considerable epitope overlap in asecond higher affinity assay. However, it isassumed that at least in the initial stages, anassay detecting the maximum numbers of posi-tive clones is required. Suitable conditions forobtaining specific responses can then beachieved later.f. Number of assays
In a typical fusion using 4 9 96 well platessome 400 samples must be assayed in a shorttime. Subcloning usually involves a similarnumber although a valuable clone may be
subcloned with more. Further, a comprehensivescreen of hybridomas should ideally involve theuse of several plates under different sets ofconditions. If the final application of the anti-body is not considered then positive samplesdetected by the first screening should be assayedby a second directed to the final application,since the number of positive clones is muchlower than the total number of clones.g. Time of assay
The time of assay, in terms of clonal growth,should be soon after clones are microscopicallyvisible and again a few days after when theclones are visible to the eye. Screening shouldcontinue for several weeks. The actual assayhowever, is influenced by the antibodyconcentration.h. pH of assay
It is expected that the most antibodies havetheir optimal reaction with antigen at physio-logical pH. It is quite possible to fail to detect agood hybridoma by screening at a single pH.The pH of tissue culture fluid in which cells aregrowing can vary almost a whole pH unit. If adefined pH is required for the final applicationthen the assay should be buffered accordinglyduring the incubation of antibody with antigen,particularly for in vivo uses. Where pH adjust-ments are necessary the nature of the buffershould be considered as the buffer componentsthemselves may affect the assay.i. Temperature of assay
Dissociation constant is the variable which ismost sensitive to temperature. The best assayconditions for hybridoma favor incubation at4 �C rather than at room temperature or higherbut ideally both should be tried unless the finaluse precludes certain temperature.j. Ionic strength of assay
There is no detailed information on the ionicstrength variations in hybridoma assays. Non-specific binding is more likely to occur at lowionic strengths. If different pH buffers are usedin the assay then all buffers used should have thesame ionic strength.
198 B. D. Lakhchaura
11.4 Cell Culture Requirementsfor Hybridomas
1. MediaTwo main types of media used for hybridoma
production are Dulbecco’s Modification of EaglesMedium (DMEM) and Rosewell Park MemorialInstitute (RPMI) medium. Media are prepared indouble distilled water, sterilized by filtrationthrough 0.2 lm membranes, and usually stored in500–1,000 ml aliquots for up to 6 weeks. Both theabove media are bicarbonate buffered with phenolred indicator. The correct color for medium isbright orange indicating a pH of 7.2.
DMEM or RPMI 1640 supplemented withhigh glucose (4.58 g/l) are popularly used forculturing myeloma cells. Glutamine (2 mM finalconcentration), antibiotics such as penicillin(100 U/ml) or streptomycin (10 lg/ml) and10 % fetal calf serum is also added. Myelomacells should be grown in the presence of8-azaguanine prior to fusion to ensure HGPRTnegative character. For fusion, cells should be inlogarithmic phase (3–8 9 106/ml).2. Sera
Fetal calf serum (FCS) is used in nearly allhybridoma work because of the low level ofcontaminating immunoglobulin. It is kept frozenat -20 �C. Horse and rabbit serum is alsosometimes used.3. Antibiotics for prevention of
contaminationThe main antibiotics used in hybridoma pro-
duction are penicillin and streptomycin. Peni-cillin inhibits the growth of most gram-positivebacteria, whereas streptomycin inhibits thegrowth of most gram-negative bacteria. Theantibiotic preparations are usually made upin 100 9 stock solutions containing 107 units ofsodium benzyl penicillin and 10 g of strepto-mycin sulfate per liter. This is filter sterilizedthrough 0.2 lm membranes and stored in 20 mlaliquots at -20 �C. Some labs use fungizone(Amphotericin B) at 2.5 lg/ml medium.
Mycoplasma contamination may be pre-vented by kanamycin (100 lg/ml), tylocine(50 lg/ml) or lincomycin, and vanomycin to
some extent. Exposure of cells at elevated tem-peratures has also been reported to curb myco-plasma contamination.
Viral contamination with Epstein Barr (EB)virus in a latent form is usually present if humanlymphocytes are used. The virus transformsculture especially in the absence of cytotoxic Tlymphocytes. Lymphocyte donors are usuallytested for antibody to the viral capsid antigen byuse of cell line P3HR1which secretes non-transforming EB.
Yeast contamination is rare. The use of fun-gizone or nystatin (50 lg/ml) is usually helpfulin such situations.4. Feeder cells
Feeder cells are absolutely essential forcloning of hybridomas. They feed or nourish theemerging hybridomas but may have a limitedlifespan. If phagocytic cells such as macro-phages and monocytes are used they can also behelpful in cleaning debris of dead cells foundafter aminopterin treatment. Spleen cells areconvenient to use as feeders.5. Important chemicals
Dimethyl sulphoxide (DMSO): DMSO isrequired for freezing and thawing of cells andsome use it in fusion procedures. It is sterilizedby autoclaving or filtration.
Polyethylene glycol (PEG): All fusionsdesigned to produce hybridomas are performedwith chemical fusogens and PEG is the mainchemical used for this.
HO CH2CH2Oð ÞnCH2CH2OH
Structure of polyethylene glycol PEGð Þ
PEG has a molecular weight range from 200 to20,000 Daltons. It is toxic to cells; low molecularweight PEG is more toxic than high molecularweight PEG. Most successful fusions are per-formed with PEG of molecular weight 600–6000Daltons. Most protocols use PEG at 37 �C. It hasbeen also shown that room temperature is supe-rior to 37 �C and that the optimum pH is 7.5.
The exact mechanism of fusion is not fullyunderstood, but it is thought that hydrophilicPEG occupies the ‘physical free space’ leading
11 Monoclonal Antibody Production and Applications 199
to agglutination of the cells. This occurs atconcentrations of PEG in the range of 40–50 %with some variations. Lower PEG concentra-tions such as 35 % can, however, be used with alonger exposure time. Mostly fusions are doneusing PEG in 15 % DMSO.6. Myeloma cells
Myeloma cells are cancerous cells and impartimmortality to hybridoma clones. Spleen cellsare antibody-producing cells; hence provide theability to generate the antibody. Most of themurine myeloma cell lines are available fromAmerican Type Culture Collection (ATCC),Maryland 20852 USA. A few commonly usedmurine myelomas are Sp 2/0 Ag.14, NS1/1, Ag4.1 NSO, and P3X63 Ag8.
Rational for using HAT media for selectinghybridoma clones is that, myeloma cells aredeficient in HGPRT and thymidine kinase (TK),hence lack the salvage pathway for nucleotidesynthesis. Aminopterin is added to inhibit the denovo pathway of nucleotide synthesis. Thusmyeloma cells do not grow in HAT medium.The spleen cells are primary cells and cease togrow after a few divisions. Only hybridoma cellswhich have acquired HGPRT and TK genesfrom spleen cells multiply and form clones.These cells secrete very high level of antibodiesin the media.7. Subcloning of hybridoma clones
To ensure monoclonality of the hybridomaclones, the cells should be re-cloned under lim-iting dilution conditions for a few cycles. Cul-ture is diluted so that 1–2 cells per well aredistributed. Limited dilution cloning techniquerequires presence of feeder cells in the cloningwells. This process eliminates non-secretors andcontaminating hybridomas ensuring monoclo-nality of the antibody.8. Enzyme Linked Immunosorbent Assay
for antibody detectionThe wells of the ELISA plate are coated with
the antigen and the supernatant from thehybridoma wells is added. The unbound anti-body is washed away. A second antibody con-jugated to an enzyme which is specific to the Fc(fragment crystallizable) region of the firstantibody is added and allowed to bind to the first
antibody. After washing, the substrate for theenzyme is added and the reaction is allowed toproceed. If the first antibody binds to the antigenthe second antibody will also bind (to the firstantibody) and the enzyme will react with thesubstrate and colored product is formed whichcan be easily visualized.
11.5 Steps in Monoclonal AntibodyProduction
1. Balb/c mice are immunized with the antigenvia suitable route. For most protein antigens,2–3 immunizations are sufficient to evoke astrong immune response. Usually a group of4–5 mice are immunized and the oneexhibiting highest titer is used as the sourceof plasma cells. This mouse is immunizedby IV (intravenous) route and after 3 daysthe mouse is sacrificed.
2. Spleen is removed from the mouse andsingle cell suspension is prepared.
3. Exponentially growing myeloma cells aremixed with splenocytes in the proportion of1:10.
4. Fusion between these cells is accomplishedin the presence of a fusion agent, usuallypoly ethylene glycol (MW 4,000). Bothcells are centrifuged and the supernatantmedia is removed. The cell bottom is loos-ened by gentle tapping and 50 % PEG isslowly added drop-wise. The cell suspen-sion is held at 37 �C for 2–3 min. After that,PEG is diluted with the serum free mediaand cells are centrifuged to remove thePEG.
5. The cell pellet is suspended in about 100 mlhypoxanthine, guanine, and aminopterin(HAT) medium. The HAT medium consistsof RPMI 1,640 medium supplemented withHAT.
6. The suspension is distributed in ten 96-wellmicrotiter plates.
7. The plates are incubated at 37 �C in a car-bon dioxide incubator overnight (the incu-bator is operated at 37 �C, 95:5 air and CO2
environment and 95 % humidity)
200 B. D. Lakhchaura
8. Next day, 0.1 ml HAT media is added toeach well.
9. Plates are examined regularly for appear-ance of growth.
10. Between 10 and 14 days of incubation,some wells exhibit growth and mediumturns slightly yellowish in color in the wells.
11. The supernatant from wells is screened forthe presence of antibodies by Enzyme-Linked Immunosorbent Assay (ELISA)technique.
12. Hybridoma cells from positive wells areexpanded by growing in 24-well plates fol-lowed by growth in 25 cm3 culture flask.
13. Desirable hybridoma clones are preservedby cryopreservation in liquid nitrogen.
11.6 Large-Scale Productionof Antibodies
Antibodies can be produced by two differenttechniques. In the first method the hybridomacells are grown in serum free media. Growingcells secrete antibodies which can be purified byaffinity chromatography technique. The antibodyyield usually varies between 10 and 50 lg/ml. Inexceptional cases one may get a yield of up to100 lg/ml as well. The second method whichinvolves production of ascitic fluid can produceup to 20 mg/ml antibody yield. For ascitic fluidproduction, the hybridoma cells are injected inthe peritoneal cavity of pristine primed-immu-nocompromised Balb/c mice. The ascitic fluiddevelops in the peritoneal cavity and can beeasily aspirated with the help of 18 gaugehypodermic needle. From ascitic fluid alsoantibodies can be purified by affinity chroma-tography using Protein A coupled Sepharosebeads. Lately, due to objection from organiza-tions for prevention of cruelty to animals, ascitesproduction is becoming problematic. Antibodiescan also be directly obtained from the fluid byammonium sulfate precipitation.
The rapid progress being made in the com-mercialization of monoclonal antibodies has ledto large-scale production of MAb. Commercialinterests consider production scales of 0.1–10 g
as small, 10–100 g as medium and over 100 g aslarge. Commercial-scale production is generallyperformed to produce MAb for three purposes:diagnosis, therapy and research, and develop-ment of new therapeutic agents. Monoclonalantibodies are being manufactured for clinicaltrials in large-scale suspension culture in fer-menters. A completely automated pilot plantused for fermentation has been employed withdirect digital control (DDC) technology formonitoring and regulating growth of humancells. A human hybridoma cell line (3D6) pro-ducing anti-human immunodeficiency virus(HIV)-1 antibodies was used as a model forlarge-scale production (300-liter airlift fermen-tor) in continuous culture. The production ofanti-a-fetoprotein monoclonal antibodies fordiagnostic use was carried out in a stirred tankfermenter equipped with a double membranestirrer for bubble free aeration and continuousmedium perfusion.
11.7 Applications of MonoclonalAntibodies
Monoclonal antibodies are widely used asdiagnostic, research, and therapeutic agents fortreatment of numerous human ailments. Theapplications can be divided in mainly threemajor groups:1. Immunodiagnostic reagent2. Research tools in molecular localization of
antigens in intact cells and epitope analysis.3. Immunotherapeutic and Immunoprophylactic
agents.
11.7.1 Immunodiagnostic Reagent
Monoclonal antibodies are useful tools fordetection of antigens in various body fluids andtissues. MAb-based commercial kits are avail-able for detection of viral, bacterial, fungal, andparasitic pathogens. MAbs attached to latexbeads or erythrocytes can be added to a bodyfluid. Presence of antigen causes agglutination.This is easily visible. It should be kept in mind
11 Monoclonal Antibody Production and Applications 201
that some antigens may not cause agglutination.Detection of antigens in intact tissue can bemade with use of either a fluorochrome-labeledor enzyme-labeled or isotope-labeled antibody.Pathogens can be visualized by fixing a tissueslice on microscope slide or slide culture of thepathogen. Detection is made either with the helpof a fluorescent microscope, use of an enzymesubstrate which provide an insoluble coloredproduct in case of fluorochrome-labeled orenzyme-labeled MAbs, respectively. A radio-isotope can be detected by autoradiography.Many animal viruses (Koprowski and Wiktor1980; Sonza et al. 1983), bacteria (Gustafsonet al. 1982), and parasites (Wrisht et al. 1983;Scott 1983) have been diagnosed using MAbs.
A very large number of monoclonal antibodieshave been produced to a wide range of virusessuch as influenza, hepatitis, polio, Epstein Barrvirus, and rabies. Their high specificity has led toaccurate identification between similar stains ofvirus such as Herpes simplex Types I and II. Theymay also be used for early diagnosis of the IgMproduction in affected patients.
Antibodies have been designed for bacterialdiseases as well though they are less common.They are helpful in analysis of bacterial sporeswhich tend to show extensive cross-reactivity inconventional immunological analysis. Mono-clonal antibodies to bacterial toxins have alsobeen generated.
The potential of monoclonal antibodies in thestudy of parasitic diseases has already beenwidely exploited for diseases such as malaria,schistosomiasis, and leishmania. The advantagehere is that the disease may be studied not onlyfor diagnostic purpose but also in greater depthsince many different surface antigens may beexpressed at varying stages of the life cycle ofthe parasite.
Competitive or noncompetitive ELISA pro-cedures have been developed for detectingantigens in body fluids. The MAb is usuallyattached to a microtiter well and body fluid(containing the antigen) is allowed to bind to the
antibody. Detection can be made by using adifferent MAb coupled to an enzyme or radio-isotope. Two MAbs used should be either fordifferent epitopes or against a repetitive epitope.In competitive ELISA, the test sample is mixedwith the known amount of labeled antigen andadded to the antibody-coated microtiter well.Less labeled antigen binds to MAb withincreasing concentrations of the test antigen.
Some precautions are required for diagnosticutility of the MAbs. Since there are multiplestrains of the particular organism which cancause infection, a MAb may detect a singlestrain only. These strains may differ in theirantigenic epitopes. Therefore, a monoclonalantibody against a common epitope must beselected for immunodiagnosis. MAbs are extre-mely useful for unambiguous diagnosis of dis-eases which produce closely related symptoms.As an example, MAb against Marek’s diseasedoes not bind to lymphoid leucosis virus thoughboth cause similar symptoms. Similarly, MAbscan distinguish canine parvovirus infection frompanleukopenia virus.
MAb are being used for cancer diagnosis too.MAbs to certain tumor markers are being usedfor detection in tissue fluid, tissue explants, or inintact organism through imaging. Precise infor-mation about the type of cancer is very helpfulin its therapy. Tumor markers against prostatecancer, colorectal cancer, and ovarian tumorshave been utilized for monitoring the tumorregression or progression as the marker levelsfluctuate with its status. MAbs are also beingextensively used for diagnosis of hematopoieticmalignancies such as primary or acquiredimmunodeficiency syndrome (AIDS).
Immunoscintigraphy procedures have beendeveloped for scanning human body for thelocation of cancerous tissue. The MAbs arelabeled with radioisotopes such as 131I (Iodine)or 99Tc (Technetium) and injected intravenouslyinto the patient. It binds to cancer cells in dif-ferent locations which can be detected byimaging.
202 B. D. Lakhchaura
11.7.2 Basic Research
The potential of monoclonal antibody in basicresearch is substantial. In principle they canresolve a single protein from a complex mixtureor a single epitope responsible for a specificfunction of a complex macromolecule. Theyhave been widely used in basic enzymology, innucleic acid structural studies, and in the anal-ysis of hormone receptors. Another field inwhich monoclonal antibodies may prove ofparticular value is in the study of chromosomalproteins which are responsible for determiningcell phenotype. For this they are ideal tools forthe dissection of the complex mixture ofproteins.
11.7.3 Therapeutic Applications
One of the earliest applications of MAb forhumans came in the form of murine MAb OKT3(orthoclone) which was used for preventingrenal-transplant rejection. Human allografts areoften rejected by the host and to sustain theallograft the patients are subjected to adminis-tration of highly toxic immunosuppressive drugsor high doses of glucocorticoids. OKT3 is anantibody which prevents the proliferation ofcytotoxic T cells, hence prevents graft rejection.
Murine MAbs are not preferred for humantherapeutic application, as this initiate immuneresponse in human patients. In order to preventor minimize deleterious immune responsesmurine MAbs have been humanized. Usinggenetic engineering techniques human Fc regionis grafted onto the variable region of murineMAb. This reduces the immune response as theFc region is strongly immunogenic. With furtherimprovement in the technology, now even theframework regions of the MAbs have beengrafted along with the Fc region from humanimmunoglobulins. Humanized murine MAbswhich have only complementary determiningregion (CDR) from the murine MAb and rest ofthe molecule is of human immunoglobulin, arefinding application mainly in cancer therapy.
HIV infects Th (T helper cell) lymphocytesvia gp 120 envelope protein of the virus. Virusbinds to Th lymphocytes on CD4+ (Cluster ofdifferentiation) receptors via gp 120 glycopro-tein. Using genetic engineering techniques, Fcregion of the antibody has been conjugated to aCD4 glycoprotein molecule. When this conju-gate is administered to HIV patients, the HIVinfected cells of the patient exhibiting gp 120bind to CD4+ Fc conjugate. This event triggerscell mediated immune response and the infectedcell is killed. This strategy may prove as aneffective therapy for HIV patients.
MAbs can also be used for targeting drugs tocancer cells or virally infected cells. The drug isconjugated onto the MAb which binds to onlyaffected cell and it is selectively delivered to thecancer cell or virally infected cell. Not onlydrugs but also deadly toxins such as diphtheriatoxin or ricin could be coupled to MAb mole-cules and can be used to selectively kill targetedcells. The normal cells are not killed by the drugor the toxin and prevent the side effects ofchemotherapy.
Anti-idiotype MAbs can replace the antigenin detection assays. Anti-idiotype antibody(Ab2) has binding ability to antigen bindingamino acid sequence (epitope) of the hyper-variable region of the antibody (Ab1). Theresulting Ab2 is capable of competing with theoriginal antigen. Thus if the antigen availabilityis limiting or toxic, the MAb can easily besubstituted for the antigen.
A substantial number of MAbs have beenapproved by U.S. Food and Drug Administrationfor use in humans. A few of these are listed here.
RiuxiMAb and Zevalin conjugated with iso-topes of indium or yttrium have been approvedfor treatment of B cell lymphomas. Both theseantibodies are targeted to CD20, a markerpresent on B cells. Administration of 131I labeledTositumoMAb in B cell lymphoma patients havekept them free from disease for a substantialnumber of years. These antibodies also bind toCD40 receptors on B cells. CetruxiMAb andHerceptin have been used for successful treat-ment of breast cancer. CetruxiMAb is specificfor HER1 (human epidermal growth factor)
11 Monoclonal Antibody Production and Applications 203
receptor present on breast cancer cells. Hercep-tin blocks the growth factor receptor HER2which is also found in some breast cancers. MAbagainst CD30 conjugated with Vedotin blocksthe proliferation of lymphomas. The preparationis called Adcetris. A number of MAb prepara-tions are also available for treatment of leuke-mia. These are Lymphocide and AlemtuzuMAb.Lymphocide is against CD22 receptor and isused for treatment of B cell leukemias. Ale-mtuzuMAb is against CD52 receptor which isfound on B as well as on T cells. Thus thisantibody is useful for treatment of both B and Tcell lymphomas. An antibody called Lym-1 hasbeen shown to bind to HLA-DR which isexpressed heavily on lymphoma cells. An anti-body LipimuMAb has shown promise in sup-pressing all types of tumors acting as tumorsuppressor agent.
Vitaxin is an antibody against integrin foundon the blood vessels of tumor cells and hasshown promise to be an inhibitor of angiogene-sis selectively in tumor cells. This results inregression of solid tumors. BevaxizuMAb pre-vents clumping of platelets and prevents re-clogging of arteries after angioplasty.
Autoimmune disorders are generally difficultto treat by using conventional drugs. Two MAbsnamely InflixiMAb and AdalimuMAb have beenused for treatment of rheumatoid arthritis andChrohan’s disease. These antibodies bind toTNF-alpha (Tumor necrosis factor) and reducethe proliferation of Th1 cells which are active inenhancing cell-mediated immune response.OmalizuMAb binds to IgE receptors and is usefulfor treatment of allergic asthma. DaclizuMAbbinds to IL-2 receptors on the surface of activatedT cells preventing acute graft rejection of thetransplanted kidney. This antibody has alsoshown promise against T cell lymphomas.
11.7.4 Monoclonal Antibodiesand Infectious Diseases
A humanized MAb against respiratory syncytialvirus has been approved for treatment of pre-mature infants or infants suffering from
bronchopulmonary dysplasia. The MAb calledPalivizuMAb has shown great promise in suchcases. Similarly, many other MAbs are undertrial for combating viral infections. MAbs arealso very useful tools for dissecting viral anti-genic epitopes which is essential for developingefficacious vaccines for viral infections.
MAbs have also found applications in eluci-dating host–parasite relationships. In order tounderstand the relationship between the host andparasites, e.g., protozoa and helminthes in dif-ferent stages of their life cycle, panels of anti-bodies have been produced against stage specificantigens. Antigens from some of the parasiteshave been fractionated using MAbs and theantigens providing protective immunity havebeen identified. These antigens could be used forvaccination against the parasite.
In case of bacterial pathogens, MAbs havebeen extremely useful tools for unambiguousdiagnosis. Bacterial antigenic epitopes are beingcharacterized with the objective of finding anti-gens with better immunogenic potential. Sero-type specific MAbs have been developed fordetection of various leptospira, causing infec-tions in humans and animals.
11.7.5 Miscellaneous Uses of MAbs
MAbs can be effectively used for purification ofantigens from a complex mixture. MAbsattached to solid particles can be used to preparea series of columns. The complex mixture isthen serially passed through different columnsand from each column a single antigen can beobtained. Thus a panel of MAbs can be used forfractionation of antigens from a cell.
MAbs are useful reagent for mapping epi-topes on complex antigen. They are also beingused for isolating different cells of immunesystem. CD4+ and CD8+ MAbs are used forseparating T helper and T cytotoxic cells. Fur-ther with appropriate MAbs TH1 and TH2 cellscan also be fractionated.
Panel of MAbs used for generating proteinmicroarrays are highly useful tools for proteincharacterization. MAbs are used for blood group
204 B. D. Lakhchaura
analysis. Using chromosome Y specific MAbs,embryos can be sexed. This is being widely usedin dairy industry for producing only femalecalves.
For passive immunization humanized MAbsare used as prophylactic agents. These can alsobe used for neutralizing pathogens in acutecases.
Monoclonal antibodies are also used in thedetection of adulteration in meat. Poultry(chicken and turkey) tissue is a major source ofprotein and less expensive than red meat, whichis consumed and imported throughout theworld. Increasing use of mechanically separatedpoultry meat have a potential for the adultera-tion of red meat by poultry products. Martin(1989) produced and characterized MAbsagainst species-specific sarcoplasmic protein ofchicken. Three MAbs are capable of distin-guishing between muscle extracts of the mostfrequently, marketed avian (chicken and turkey)and mammalian (beef, pork, horse and lamb)species of meat animals. One of these anti-bodies has the added advantage of distin-guishing between chicken and turkey extractsby ELISA. MAb technology will improvediagnosis and serotyping/pathotyping proce-dures in poultry disease management.
11.8 Conclusion
Monoclonal antibodies are antibodies having asingle specificity and are continuously secretedby ‘‘immortalized’’ hybridoma cell, which is abiologically constructed hybrid between anantibody-producing, mortal, lymphoid cell, and
a malignant, or ‘‘immortal’’, myeloma cell. MAbare more pure than the polyclonal antibodies andbecause of their specificity, are used in bio-medical research, in diagnosis of diseases, and intreatment of infections and cancer. These havealso been used for animal disease diagnosis ofpoultry diseases, infectious bronchitis, and avianinfluenza. Commercialization of MAb has led totheir large-scale production in fermentors.
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11 Monoclonal Antibody Production and Applications 205
12Edible Vaccines
Jyoti Saxena and Shweta Rawat
Abstract
In recent years edible vaccine emerged as a new concept developed bybiotechnologists. Edible vaccines are subunit vaccines where the selectedgenes are introduced into the plants and the transgenic plant is theninduced to manufacture the encoded protein. Foods under such applica-tion include potato, banana, lettuce, corn, soybean, rice, and legumes.They are easy to administer, easy to store and readily acceptable deliverysystem for different age group patients yet cost effective. Edible vaccinespresent exciting possibilities for significantly reducing various diseasessuch as measles, hepatitis B, cholera, diarrhea, etc., mainly in developingcountries. However, various technical and regulatory challenges need toovercome in the path of this emerging vaccine technology to make ediblevaccine more efficient and applicable. This chapter attempts to discusskey aspects of edible vaccines like host plants, production, mechanism ofaction, advantages and limitations, applications, and different regulatoryissues concerned to edible vaccines.
12.1 Introduction
Vaccine is a biological preparation intended toproduce immunity to a disease by stimulatingthe production of antibodies. Dead or attenuatedorganisms or purified products derived from
them are generally used to produce variousvaccines. Over the past decade, scientificadvances in genetics, molecular biology, andplant biotechnology have improved the under-standing of many infectious diseases and led tothe development of vaccination programs. Themost common method of administering vaccinesis by injection but some are given by mouth ornasal spray. Though immunization is the safestmethod to combat the diseases worldwide butthere are many constraints regarding its mode ofproduction, distribution, delivery, cost, and lackof enough research. Hence it is desirable to lookfor an effectual and powerful yet cost effective,easy for storage and distribution yet safe methodof immunization. It should also be readily
J. Saxena (&) � S. RawatBiochemical Engineering Department, BT KumaonInstitute of Technology, Dwarahat, Uttarakhand263653, Indiae-mail: [email protected]
S. Rawate-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_12, � Springer India 2014
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acceptable to all sociocultural groups around theglobe. Research underway is dedicated to solv-ing these problems by finding ways to produceedible vaccines in the form of transgenic plantswhich have been investigated as an alternativemeans to produce and deliver vaccine.
Edible vaccines are called by several alter-native names such as food vaccines, oral vac-cines, subunit vaccines, and green vaccines.They seem to be a viable alternative especiallyfor the poor and developing countries. They havecome up as great boon in medicinal science forwhich biotechnologists should be given allcredit. The concept of edible vaccines lies inconverting the edible food into potential vaccinesto prevent infectious diseases. It involves intro-duction of selected desired genes into plants andthen inducing these altered plants to manufacturethe encoded proteins. It has also found applica-tion in prevention of autoimmune diseases, birthcontrol, cancer therapy, etc. Edible vaccines arecurrently being developed for a number ofhuman and animal diseases. This new technologyhopefully will contribute positively toward theglobal vaccine programs and have a dramaticimpact on health care in developing countries.
12.2 Historical Background
Many people in developing countries do nothave access to the vaccines they need, as thetraditional vaccines are costly and require skilledmedical people for administration and are lesseffective in inducing mucosal immune response.It was these needs which inspired Hiatt et al.(1989) who attempted to produce antibodies inplants which could serve the purpose of passiveimmunization. The first report of edible vaccine(a surface protein from Streptococcus) intobacco, at 0.02 % of total leaf protein level,appeared in 1990 in the form of a patent appli-cation published under the international patentcooperation treaty. By conceiving the idea ofedible vaccine Dr. Charles Arntzen tried torealize it (Arntzen 1997). In 1992, Arntzen andcoworkers introduced the concept of transgenicplants as a production and delivery system for
subunit vaccines in which edible tissues oftransgenic crop plants were used (Mor et al.1998). They found that this concept couldovercome the limitations of traditional vaccines,thereby triggering the research on edible vac-cine. In 1990s, Streptococcus mutans surfaceprotein antigen A was expressed for the firsttime in tobacco. The same group also pioneeredthe field with work on hepatitis B and heat-labile toxin, B subunit in tobacco plants andpotato tubers. In the same year, the successfulexpression of hepatitis B surface antigen(HBsAg) in tobacco plants was also achieved(Mason et al. 1992). To prove that plant-derivedHBsAg could stimulate mucosal immuneresponses via oral route, potato tubers were usedas an expression system and were optimized toincrease the accumulation of the protein in planttubers (Richter et al. 2000).
Parallel to the evaluation of plant-derivedHBsAg, Mason and Arntzen explored plantexpression of other vaccine candidates includingthe labile toxin B subunit (LT-B) of enterotox-igenic Escherichia coli (ETEC) and the capsidprotein of Norwalk virus. The plant-derivedproteins correctly assembled into functionaloligomers that could elicit the expected immuneresponses when given orally to animals (Masonet al. 1998).
In 1998 a new era was opened in vaccinedelivery when researchers supported by theNational Institute of allergy and infectious dis-eases (NIAID) have shown for the first time thatan edible vaccine can safely generate significantimmune responses in people. The report bycollaborators from the University of Maryland inBaltimore, the Boyce Thompson Institute forPlant Research in Ithaca, N.Y., and TulaneUniversity in New Orleans appeared in the Mayissue of Nature Medicine. According to the thenDirector of NIAID ‘‘Edible vaccines offerexciting possibilities for significantly reducingthe burden of diseases like hepatitis and diar-rhea, particularly in the developing world wherestoring and administering vaccines are oftenmajor problems,’’ Mor et al. (1999) also dis-cussed the rapid increase of research in theedible-vaccine field and pointed out that plants
208 J. Saxena and S. Rawat
could be used to create multicomponent vac-cines that can protect against several pathogensat once. This is an aspect of the edible-vaccineapproach that further strengthens its impact.Later, in 2003 Sala and research group reportedthat proteins produced in these plants inducedthe mucosal immune response which was themain aim behind this concept.
Research into edible vaccine is still at a veryearly stage and scientists have a long way to gobefore it will become a major part of immuni-zation program world wide.
12.3 Choice of Host Plantfor Edible Vaccine
To date, many plant species have been used forvaccine production. The choice of the plantspecies is important. An edible, palatable plant isnecessary if the vaccine is planned for raw con-sumption. In case of vaccine for animal use, theplant should preferentially be selected amongthose consumed as normal component of theanimal’s diet. Some food vehicles are discussedbelow:
12.3.1 Tobacco
The concept of edible vaccine got impetus afterArntzen and coworkers expressed HBsAg intobacco. The first edible vaccine was produced intobacco in 1990 in which 0.02 % recombinantprotein (a surface protein from Streptococcus) ofthe total soluble leaf proteins was found. Itappeared in the form of a patent applicationpublished under the International Patent Cooper-ation Treaty. Transgenic tobacco is successfully
engineered for the production of edible vaccinesagainst hepatitis B antigen using ‘s’ gene ofhepatitis B virus (HBV). The optimum level ofrecombinant protein was obtained in leaves andseeds. Since acute watery diarrhea is caused byenterotoxigenic E. coli and Vibrio cholerae thatcolonize the small intestine and produce one ormore enterotoxin, an attempt was made towardthe production of edible vaccine by expressingheat-labile enterotoxin (LT-B) in tobacco.Besides, antibodies against dental caries, expres-sed in tobacco, are already in preclinical humantrials. Italian researchers have now developed animmunologically active, cost-efficient vaccineagainst human papilloma viruses (HPV). HPV arethe causative agents for cervical cancer, and arealso involved in skin, head, and neck tumors.Cervical cancer is one of the main causes ofcancer-related deaths.
12.3.2 Potato
Genetically modified potatoes are also a viableoption and seem to be the desired vector. Many ofthe first edible vaccines were synthesized inpotato plants. The transgenic potatoes weredeveloped and grown by Arntzen and Mason andtheir research group at the Boyce ThompsonInstitute for Plant Research, Cornell University.Previously, NIAID supported in vitro and pre-clinical studies by John Clements and colleaguesat Tulane University School of Medicine, inwhich 14 volunteers ate bite-sized pieces of rawpotato that had been genetically engineered toproduce part of the toxin secreted by E. colicausing diarrhea. The investigators periodicallycollected blood and stool samples from the vol-unteers to evaluate the vaccine’s ability to stim-ulate both systemic and intestinal immuneresponses. Ten of the 11 volunteers (91 %) whoingested the transgenic potatoes had fourfold rise
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in serum antibodies at some point after immuni-zation, and 6 of the 11 (55 %) also showed four-fold mount in intestinal antibodies. The potatoeswere well tolerated and no one experienced seri-ous adverse side effects. Vaccine developmenthas successfully tested a potato-based vaccine tocombat the Norwalk Virus, which is spread bycontaminated food and water. The virus causessevere abdominal pain and diarrhea.
A research team led by William Langridge ofthe Loma Linda University in California hasreported that transgenic potatoes engineered with acholera antigen, CTB can effectively immunizemice. Mice fed transgenic potatoes produce chol-era-specific antibodies in their serum and intestine;IgA and IgG antibodies reach their highest levelsafter the fourth feeding. In yet another experimentgenetically engineered potatoes containing ahepatitis B vaccine have successfully boostedimmunity in their first human trials.
Attempts have also been made to boil thepotatoes as raw potatoes are not very appetizingbut unfortunately the cooking process breaksdown about 50 % of the proteins in the vaccine.While some proteins are more tolerant to heat,for most proteins it will be necessary to amplifythe amount of protein in the engineered foods ifthey are to be cooked before consumption.
12.3.3 Tomato
Tomatoes are an excellent candidate becausethey are easy to manipulate genetically and newcrops can be grown quickly. Moreover, they arepalatable and can be eaten raw. While tomatoesdo not grow well in the regions in which theedible vaccines are most needed, the engineeredtomatoes can be dried or made into a paste tofacilitate their delivery.
The anti-malaria edible vaccines in differenttransgenic tomato plants expressing antigenictype(s) have been proposed by Chowdhury andBagasara in 2007. They hypothesized thatimmunizing individuals against 2–3 antigensand against each stage of the life cycle of themultistage parasites would be an efficient,inexpensive and safe way of vaccination.Tomatoes with varying sizes, shapes, and colorscarrying different antigens would make thevaccines easily identifiable by lay individuals.
Tomatoes serve as an ideal candidate for theHIV antigen because they unlike other trans-genic plants that carry the protein, are edible andimmune to any thermal process, which help toretain their healing capabilities. Scientists haveclaimed that tomatoes could be used as a vaccineagainst Alzheimer’s disease. The work is inprogress to genetically modify the fruit to createan edible vaccine that fires up the immune sys-tem to tackle the disease by attacking the toxicbeta-amyloid protein that destroys vital connec-tions between brain cells, causing Alzheimer’s.
Researchers have engineered tomato plants(Lycopersicon esculentum Mill var. UC82b) toexpress a gene for the glycoprotein (G-protein),that coats the outer surface of the rabies virus.The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus,including the signal peptide, under the control ofthe 35S promoter of cauliflower mosaic virus(CaMV).
12.3.4 Banana
A common fruit—the banana—is currentlybeing considered as a potential vehicle for vac-cines against serious as well as too commondiseases. The advantage of bananas is that they
210 J. Saxena and S. Rawat
can be eaten raw as compared to potatoes or ricethat need to be cooked and can also be con-sumed in a pure form. Furthermore, childrentend to like banana and the plants grow well inthe tropical areas in which the vaccines areneeded the most. Hence, the research is leaningtoward the use of banana as the vector since alarge number of third-world countries, whowould benefit the most from edible vaccineshave tropical climates. On the negative side, anew crop of banana plants takes about12 months to bear fruit. After fruiting, the plantsare cut down and a new crop of vaccine-bearingplants must be planted.
Researchers have also developed bananas thatdeliver a vaccine for HBV. The banana vaccineis expected to cost just 2 cents a dose, as com-pared to the $125 for the currently availableinjectable vaccine.
12.3.5 Maize
Maize has also been used as a vector for variousedible vaccines. Egyptian scientists have genet-ically engineered the maize plants to produce aprotein known as HbsAg which elicits animmune response against the hepatitis B virusand could be used as a vaccine. If human trialsare successful more than 2 billion peopleinfected with hepatitis B, and about 350 millionof these at high risk of serious illness and death
from liver damage and liver cancer would bebenefited.
Researches are in offing at Iowa State Uni-versity with the aim to allow pigs and humans toget a flu vaccination simply by eating corn orcorn products. It is quite likely that corn vaccinewould work in humans when they eat corn oreven corn flakes, corn chips, tortillas, or any-thing that contains corn.
Genetically modified maize could provideprotection to chickens against a highly conta-gious and fatal viral disease affecting mostspecies of birds. Mexican researcher OctavioGuerrero-Andrade and his colleagues at theCentre for Research and Advanced Studies inGuanajuato, Central Mexico, genetically modi-fied maize to create an edible vaccine againstNewcastle disease virus (NDV). They inserted agene from the NDV, a major killer of poultry indeveloping countries, into the maize DNA andfound antibodies against the virus in chickensthat ate the genetically modified maize. One pigvaccine has also been produced in cornsuccessfully.
Efforts are being made by US companyProdiGene to genetically modify maize to con-tain a key protein found on the surface of themonkey form of HIV. According to US NationalInstitute of Health this development brings anedible, more effective, HIV vaccine for people astep closer.
Transgenic maize expressing the rabies virusglycoprotein (G) of the Vnukovo strain has alsobeen produced using ubiquitin maize promoterfused to the whole coding region of the rabiesvirus G gene, and a constitutive promoter fromCaMV. Maize embryogenic callus were trans-formed with the above construct by biolistics.Regenerated maize plants were recovered andgrown in a greenhouse. The amount of G-proteindetected in the grains was approximately 1 % ofthe total soluble plant protein.
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12.3.6 Rice
Rice is another potential crop which has beenused for developing vaccines. It offers severaladvantages over traditional vaccines; it does notrequire refrigeration. In fact, the rice proved justas potent after 18 months of storage at roomtemperature and the vaccine did not dissolvewhen exposed to stomach acids. In an attempt,predominant T cell epitope peptides, which werederived from Japanese cedar pollen allergens,were specifically expressed in rice seeds anddelivered to the mucosal immune system (MIS);the development of an allergic immune responseof the allergen-specific Th2 cell was suppressed.Furthermore, not only the specific IgE produc-tion and release of histamine from mast cellswere suppressed, but the inflammatory symp-toms of pollinosis, such as sneezing, were alsosuppressed. These results suggest the feasibilityof using an oral immunotherapy agent derivedfrom transgenic plants that accumulate T cellepitope peptides of allergens for allergytreatment.
The transfer of genetic material from themicrobe responsible for producing cholera toxininto a rice plant has been achieved. The plantsproduced the toxin and when the rice grainswere fed to mice they provoked immunity fromthe diarrhea-causing bacterium.
12.3.7 Spinach
Genetically modified spinach has also beenconsidered for the development of edible vac-cine. Spinach is being investigated as a plant-derived, edible vehicle for anthrax vaccine, aswell as a vehicle for the HIV-1 Tat protein (aprospective vaccine candidate). In an experi-ment a fragment of protective antigen (PA) thatrepresents most of the receptor-binding domainwas expressed as a translational fusion with acapsid protein on the outer surface of tobaccomosaic virus, and spinach was inoculated withthe recombinant virus. The plant-expressed PAis highly immunogenic in laboratory animals.
Among other food crops with potential to bedeveloped as edible vaccine; sweet potato, pea-nuts, lettuce, watermelon, and carrots are on thetop priority. The development of plant-basedvaccines to protect against many other diseases,such as HIV-1, hepatitis B, rabies, and non-Hodgkin’s lymphoma are ongoing throughoutthe globe using one of these edible plants.
The advantages and disadvantages of variousplant host systems are given in Table 12.1.
12.4 Advantages
Conventional subunit vaccines are expensiveand technology-intensive, need purification,require refrigeration, and produce poor mucosalresponse. In contrast, edible vaccines would
212 J. Saxena and S. Rawat
enhance compliance, especially in children, andbecause of oral administration would eliminatethe need for trained medical personnel. Theirproduction is highly efficient and can be easilyscaled up. For example, hepatitis B antigenrequired to vaccinate whole of China annually,could be grown on a 40-acre plot and all babies inthe world each year on just 200 acres of land. Theyare cheaper, sidestepping demands for purifica-tion (single dose of hepatitis B vaccine would costapproximately 0.43 cents), grown locally usingstandard methods and do not require capital-intensive pharmaceutical manufacturing facili-ties. Mass-indefinite production would alsodecrease dependence on foreign supply. Fear ofcontamination with animal viruses—like the madcow disease, which is a threat in vaccines manu-factured from cultured mammalian cells, iseliminated as plant viruses do not infect humans.
Edible vaccines activate both mucosal andsystemic immunity, as they come in contact withthe digestive tract lining which is not possible
with subunit vaccines which provide poormucosal response. This dual effect of ediblevaccines provides first-line defense againstpathogens invading through mucosa, such asMycobacterium tuberculosis and agents causingdiarrhea, pneumonia, STDs, HIV, etc.
The specific advantages are stated below:1. Edible means of administration.2. No need of medical personnel and syringes.3. Sterile injection conditions are no more
required.4. Economical in mass production by breeding
compared to an animal system.5. Easy for administration and transportation.6. Effective maintenance of vaccine activity by
controlling the temperature in plantcultivation.
7. Therapeutic proteins are free of pathogensand toxins.
8. Storage near the site of use.9. Heat stable, thus eliminating the need of
refrigeration.
Table 12.1 Features of different plant host systems
Plant Advantages Disadvantages
Tobacco Facile and efficient transformation system;abundant material for protein characterization
Toxic alkaloids incompatible with oral delivery;potential for outcrossing in field
Banana Cultivated widely in developing countries wherevaccines are needed; eaten raw by infants andadults; clonally propagated; low potential foroutcrossing in field; once established, plentifuland inexpensive fruits are available on a10–12 month cycle
Inefficient transformation system; little dataavailable on gene expression, especially for fruitspecific promoters; high cultivation spacerequirement; very expensive in greenhouse
Potato Facile and efficient transformation system; tuberis edible raw though not palatable; tuber-specificpromoters available; microtuber production forquick assay; clonally propagated, low potentialfor outcrossing in field; Industrial tuberprocessing well established
Relatively low tuber protein content; unpalatablein raw form; cooking might cause denaturationand poor immunogenicity of vaccine
Tomato Relatively efficient transformation system; fruit isedible raw; fruit specific promoters available;crossing possible to stack antigen genes;industrial greenhouse culture and industrial fruitprocessing well established
Relatively low fruit protein content; acidic fruitmay be incompatible with some antigens or fordelivery to infants; no in vitro system to test fruitexpression
Legumes Production technology widely established; highprotein content in seeds; stable protein in storedseeds; well suited for animal vaccines; industrialseed processing well established
Inefficient transformation systems; heating orcooking for human use might cause denaturationand poor immunogenicity of vaccine; potentialfor outcrossing in field for some species
Alfalfa Relatively efficient transformation system; highprotein content in leaves; leaves edible uncooked
Potential for outcrossing in field; deep rootsystem problematic for cleaning field
(Mason et al. 2002)
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10. Antigen protection through bioencapsulation.11. Subunit vaccine (not attenuated vaccine)
means improved safety.12. Seroconversion in the presence of maternal
antibodies.13. Generation of systemic and mucosal
immunity.14. Enhanced compliance (especially in
children).15. Delivery of multiple antigens.16. Integration with other vaccine approaches.17. Plant-derived antigens assemble spontane-
ously into oligomers and into virus likeparticles.
18. No serious side effect problems have beennoticed until now.
19. Reduced risk of anaphylactic side effectsfrom edible vaccine over injection system isone benefit reported by the Bio-Medi-cine.org. They reported that the edible vac-cine carries only part of the allergencompared to injection methods whichreduce anaphylactic risk.
20. Administration of edible vaccines to moth-ers to immunize the fetus-in utero by trans-placental transfer of maternal antibodies orthe infant through breast milk. Edible vac-cines have a potential role in protectinginfants against diseases like group-B Strep-tococcus, respiratory syncytial virus (RSV),etc., which is under investigation.
21. Edible vaccines would also be suitableagainst neglected/less common diseases likedengue, hookworm, rabies, etc. They maybe integrated with other vaccine approachesand multiple antigens may also be delivered.
12.5 Limitations and Challenges
With advancement come many hurdles andproblems, so is true for edible vaccines. Like,one could develop immunotolerance to thevaccine peptide or protein, though a littleresearch has been done on it. One of the keygoals of the edible-vaccine pioneers was to
reduce immunization costs but later many limi-tations were reported as given below:
1. Consistency of dosage from fruit to fruit,plant to plant, lot to lot, and generation togeneration is not similar.
2. Stability of vaccine in fruit is not known.3. Evaluation of dosage requirement is tedious.4. Selection of best plant is difficult.5. Certain foods like potatoes are generally not
eaten raw and cooking the food mightweaken the medicine present in it.
6. Not convenient for infants as they might spitit, eat a part or eat it all, and throw it uplater. Concentrating the vaccine into a tea-spoon of baby food may be more practicalthan administering it in a whole fruit.
7. There is always possibility of sideeffectsdue to the interaction between the vaccineand the vehicle.
8. People could ingest too much of the vac-cine, which could be toxic, or too little,which could lead to disease outbreaksamong populations believed to be immune.
9. A concern with oral vaccines is the degra-dation of protein components in the stomachdue to low pH and gastric enzymes. How-ever, the degradation can be compensatedby repeating the exposure of the antigenuntil immunological tolerance is accom-plished (Mason et al. 2002).
10. Potential risk of spreading the disease byedible vaccine delivery is a concern ofmany. Potential contamination of the oraldelivery system is a possible danger.
Foreign proteins in plants accumulate in lowamounts (0.01–2 % of total protein) and are lessimmunogenic, therefore the oral dose farexceeds the intranasal/parenteral dose. Forexample oral hepatitis B dose is 10–100 timesthe parenteral dose and 100 g potato expressingB subunit of labile toxin of ETEC (LT-B) isrequired in three different doses to be immuno-genic. Attempts at boosting the amount of anti-gens often lead to stunted growth of plants andreduced tuber/fruit formation as too muchmRNA from the transgene causes gene silencingin plant genome.
214 J. Saxena and S. Rawat
Techniques to overcome these limitations aregiven below:1. Optimization of coding sequence of bacterial/
viral genes for expression as plant nucleargenes
2. Expression in plasmids3. Plant viruses expressing foreign genes4. Coat-protein fusions5. Viral-assisted expression in transgenic plants6. Promoter elements of bean yellow dwarf
virus with reporter genes GUS (b -glucuron-idase) and green fluorescent protein (GFP),substituted later with target antigen genes.
7. Antigen genes may be linked with regulatoryelements which switch on the genes morereadily or do so only at selected times (afterthe plant is nearly fully grown) or only in itsedible regions. Exposure to some outsideactivator molecule may also be tried.
12.6 Side Effects
Development of edible vaccines is a possiblehigh-volume, low-cost delivery system for third-world countries to fight against fatal maladieslike AIDS, hepatitis, and diarrhea. Researchesby the NIAID and the University of Marylandshowed no significant side effects in a smallstudy using genetically engineered potatoes tomake toxin of the E. coli, a diarrhea-causingbacterium. Volunteers reported no seriousadverse reactions to genetically altered potatoesused to deliver edible vaccine toxin, accordingto the National Institutes of health (NIH). TheNIH reported that 10–11 volunteers who ate theraw potato bites developed four times the anti-bodies against E. coli without obvious sideeffects.
Long-term reactions to edible vaccines areyet to be determined and possible delayedreactions have not yet been discovered. Anorganized large scale study is required beforeedible vaccines are put into large scaleproduction.
12.7 Production of Edible Vaccines
Creating edible vaccines involves the geneticengineering approach for the introduction ofselected desired genes into plants and theninducing these altered plants to manufacture theencoded proteins. This process is known as‘‘transformation’’ and the plants altered withnew characteristics are called ‘‘transgenicplants’’. Like conventional subunit vaccines,edible vaccines are composed of antigenic pro-teins and are devoid of genes responsible forpathogenicity. Thus, they have no way ofestablishing infection, assuring its safety, espe-cially in immune-compromised patients.
The gene which codes the active antigenicprotein is first isolated from the pathogen and isincorporated in a suitable ‘‘gene vehicle’’. Thisgene vehicle is integrated into the genome of theplant and is allowed to express the correspond-ing antigen. Then these plant parts are fed toanimals and humans to run their course.
Two main strategies are used for the pro-duction of candidate vaccine antigen in planttissues (Fig. 12.1 a, b).1. Stable genomic integration: This is the most
popular method used for the published ediblevaccine clinical trials to date. Under thismethod the genetic line is produced that canbe propagated either by vegetative (stemcuttings) or sexual (seeds) reproductionmethods. The stable expression strategy pro-vides an opportunity to introduce more thanone gene for possible multicomponent vac-cine production. Furthermore, the choice ofgenetic regulatory elements allows organ andtissue-specific expression of foreign antigens.Stable transformation causes the desired geneto be incorporated either in nucleus or chlo-roplast. Agrobacterium mediated gene trans-fer is used for transforming the plants inwhich the gene is integrated in nucleus.Besides, direct delivery of DNA into the tis-sue can also be applied, biolistic being themost popular method. However, chloroplast
12 Edible Vaccines 215
engineering has also got impetus in lastdecade due to its various advantages overnuclear engineering.
2. Transient expression using viral vectors: Inthis method viral vectors are used as a tool todeliver genetic material into cells. A recom-binant plant virus is selected that can carrythe vaccine gene and can cause the plant toexpress the antigen by systemic infection. Ascompared to stable expression, transientexpression is difficult to initiate, because theviral vectors must be inoculated into indi-vidual host plants, but gives higher level of
expression as it allows the virus to replicateand amplify the gene copy number.Some of the most popularly used techniques
are described below:
12.7.1 Agrobacterium tumefaciensMediated Gene TransferMethod
Plant transformation mediated by the plantpathogen, A. tumefaciens has become the mostpopular method lately. It is a naturally occurring
Fig. 12.1 (a) Stable and (b) transient strategies for the production of candidate vaccine in potato and tobacco planttissue, respectively
216 J. Saxena and S. Rawat
gram-negative soil bacterium, which infects thewound sites in dicot plants causing the formationof the crown gall tumor. This bacterium iscapable of transferring a particular DNA seg-ment (T-DNA) of the tumor-inducing (Ti) plas-mid into the nucleus of infected cells where it issubsequently integrated into the host genomeand transcribed. The T-DNA usually containscancer-causing oncogenic genes and genes thatsynthesize opines which are excreted by infectedcrown gall cells and are a food source for bac-terium. During the genetic manipulation, the Tiplasmid is engineered to carry the desired genefor vaccine and the virulent genes that causetumor growth in plants are deleted. The trans-gene is integrated, expressed, and inherited inmendelian fashion. The whole plant can be thenregenerated from individual transformed plantcell. It has been studied that genes are success-fully expressed in experimental model plantsand when given orally to animals, the extract oftransgenic plant containing the antigen inducedserum antibodies, thus can be used to producethe edible vaccine.
The application of Agrobacterium mediatedtransformation is at present possible to mostspecies of agronomic interest, including mem-bers of family Graminae and Leguminosae. Thisopens interesting new aspect for the develop-ment of edible vaccines for both human andveterinary uses.
12.7.2 Biolistic Method
The second approach for nuclear transformationis based on the microprojectile bombardmentmethod, also known as the gene gun or biolisticmethod. This method is especially beneficial forthose plants which can not be transformed byA. tumefaciens mediated gene transfer method.Selected DNA sequences are precipitated ontometal microparticles and bombarded with aparticle gun at an accelerated speed in a partialvacuum against the plant tissue placed within theacceleration path. Microparticles penetrate thewalls and release the exogenous DNA inside the
cell where it will be integrated in the nucleargenome. Thus, this method effectively intro-duces DNA. The cells that take up the desiredDNA, are identified through the use of a markergene (in plants the use of GUS is most com-mon), and then cultured to replicate the gene andpossibly cloned. This method has variousadvantages including (1) thousands of particlesare accelerated at the same time causing multi-ple hits resulting in transferring of genes intomany cells simultaneously, (2) since intact cellscan be used, the difficulties encountered with theuse of protoplast are automatically circum-vented, and (3) the method is universal in itsapplication so that cell type, size, and shape orthe presence/absence of cell wall do not signif-icantly alter its effectiveness.
Another important use of the gene guninvolves the transformation of organelles such aschloroplasts, and yeast mitochondria. The bio-listic particle delivery system ‘‘shoots’’ ade-quately processed DNA particles, whichpenetrate into the chloroplast and integrate withits genome.
12.7.3 Chloroplast Transformation
The chloroplast’s transformation is an interest-ing alternative to nuclear transformation whichhas come up in recent past. All plant cells havechloroplasts that capture light energy from thesun to produce free energy through a processcalled photosynthesis. In chloroplast geneticengineering, the recombinant DNA plasmid isbound to small gold nanoparticles that areinjected into the chloroplasts of the leaf using agene gun as described above. This device useshigh pressure to insert the plasmid coated par-ticles into the cells. These plasmids containmultiple genes of importance such as the thera-peutic gene, a marker gene (may or may not befor antibiotic resistance), a gene that enhancesthe translation of therapeutic gene and two tar-geting sequences that flank the foreign gene. Theforeign genes are inserted through homologousrecombination via flanking sequences at a
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precise and predetermined location in the orga-nelle genome. The gene expression level inplastids is predominately determined by pro-moter and 50-untranslated regions (50-UTR ele-ments) (Gruissem and Tonkyn 1993). Therefore,suitable 50-UTRs including a ribosomal bindingsite (RBS) are important elements of plastidexpression vectors (Eibl et al. 1999). In order toobtain high-level protein accumulation fromexpression of the transgene, the first requirementis a strong promoter to ensure high levels ofmRNA. Most laboratories use the strong plastidrRNA operon (rrn) promoter (Prrn).
Besides gene gun, PEG mediated transfor-mation and Galistan Expansion Femto Syringemicroinjection techniques are also used for genedelivery in chloroplast. Some of the advantagesof chloroplast transformation technology are itslow cost, natural gene containment, site specificinsertion, very high level of stable expression,generation of production lines with a competi-tive timeline, elimination of gene silencing, andhigh accumulation of the recombinant protein.Precise steps are given below:
Step 3:A heteroplasmic diploid plant cell (First
round of selection)A homoplasmic diploid plant cell (Second
round of selection)Step 4:Multiple gene expressionStep 5:Reproductive organsDisintegration of paternal plastidsStep 6:Maternal inheritance of transgenic traits.
12.8 Mechanism of Action
The most common entry point for pathogens isat mucosal epithelia lining the gastrointestinal,respiratory, and urino-reproductive tracts, whichare collectively the largest immunologicallyactive tissue in body. The MIS is the first line ofdefense and the most effective site for vaccina-tion against those pathogens; nasal, and oralvaccines being the most effective. The goal oforal vaccine is to stimulate both mucosal andhumoral immunity against pathogens.
Edible vaccines have plant parts which arefed directly and the outer tough wall of plant cellacts to protect the antigens against attack by
Step 1: Single gene
Chloroplast transformation vectors
Multiple gene
Step 2:
with betaine aldehyde Higher transformation efficiency
Gene delivery
with antibiotic selection Lower transformation efficiency
(Selection) (Regeneration)
218 J. Saxena and S. Rawat
enzymes, and gastric and intestinal secretions.This method is known as bioencapsulation.Therefore, the majority of the plant cell degra-dation occurs in the intestine as a result of actionon digestive or bacterial enzymes. The antigensthus released are taken up by M cells in theintestinal lining that are present over the Peyer’spatches (PP) in the ileum and the gut-associatedlymphoid tissue. PP are an enriched source ofIgA producing plasma cells and populatemucosal tissue and serves as mucosal immuneeffectors sites. The breakdown of edible vaccineoccurs near PP, consisting of 30–40 lymphoidnodules on the outer surface of the intestine andcontain follicles from which germinal centerdevelops upon antigenic stimulation. These fol-licles act as the sites from which antigen pene-trates the intestinal epithelium, therebyaccumulating antigen within organized lym-phoid structure. The antigens then come incontact with M cells which in turn express classII MHC molecules. Antigens transported acrossthe mucous membrane by M cells can activateB-cells within these lymphoid follicles. Theactivated B-cells leave the lymphoid folliclesand migrate to diffuse mucosal associated lym-phoid tissue (MALT) where they differentiateinto plasma cells that secrete the IgA class ofantibodies. These IgA antibodies are transportedacross the epithelial cells into secretions of thelumen where they can interact with antigenspresent in the lumen and immediately neutralizethe infectious agent. The induction of mucosalimmunity by edible vaccine is depicted in theflow diagram (Fig. 12.2).
12.8.1 How Edible Vaccine ProvidesProtection?
An antigen in a food vaccine is taken up by Mcells in the intestine and passed to variousimmune system cells, which then start a defen-sive attack, as antigen is a true infectious agent,not just part of one. The response leaves long-lasting ‘‘memory cells’’ able to promptly neu-tralize the real infectious agent. The whole
procedure can be explained in two stages(Fig. 12.3 a, b) Antibodies and antibody frag-ments produced against specific antigens aregiven in Table 12.2.
12.9 Applications
There are numerous therapeutic and diagnosticapplications of edible vaccines which are sum-marized in Table 12.3. Some of the diseaseson which the work is going on are describedbelow:
12.9.1 Malaria
Malaria is a disease of humans transmitted bythe bite of an infected mosquito. It remains oneof the most significant causes of human mor-bidity and mortality worldwide. According toWHO’s 2010 world malaria report there aremore than 225 million cases of malaria killingaround 781,000 people. Three antigens are cur-rently being investigated for the development ofa plant-based malaria vaccine, merozoite surfaceprotein (MSP) 4, MSP 5 from Plasmodium fal-ciparum and MSP 4/5 from P. yoelli. Wang et al.(2004) have demonstrated that oral immuniza-tion of mice with recombinant MSP 4, MSP 4/5,and MSP 1, co-administered with CTB as amucosal adjuvant, induces antibody responseseffective against blood stage parasite.
12.9.2 Measles
Measles is an infection of the respiratory systemcaused by a virus. In an experiment mice fedwith tobacco expressing MV-H (measles virushaemagglutinin from Edmonston strain) anti-body titers five times the level considered pro-tective for humans could be attained andsecretory IgA was found in their feces. Primeboost strategy by combining parenteral andsubsequent oral MV-H boosters could inducetiters 20 times the human protective levels.
12 Edible Vaccines 219
These titers were significantly greater than witheither of the vaccine administered alone. MV-Hedible vaccine does not cause atypical measles,which may be occasionally seen with the currentvaccine. Thus, it may prove better for achievingits eradication. The success in mice has
prompted similar experiments in primates.Transgenic rice, and lettuce, and baby foodagainst measles are also being developed. Whengiven with CTB (adjuvant), 35–50 g MV-Hlettuce is enough; however, an increased dosewould be required if given alone.
Fed directly
Bio-encapsulation
AntigensReleased
Intestinal lining over Peyer’s patches/gut associated lymphoid tissue
antigenic stimulation
Antigen penetrates the intestinal epithelium
Antigen contacts M cells
Antigen transported across themucous membrane by M cells
Edible vaccine/ Plant parts
Plant cell degradation by digestive/ bacterial enzyme in intestine
Taken up by M cells
Break down of edible vaccine near peyer’s patches
Follicles develop germinal centre
Antigen accumulation within organized lymphoid
structure
Activate B cells within lymphoid follicles
Differentiate into Plasma cells
Transported to Lumen Interaction with antigen
Neutralize the infectious agent
M cells express classII MHC
Migration to MALTIg A production in plasma cells
Plant cell protect the antigen against
enzymatic attack
Fig. 12.2 Induction of mucosal immunity
220 J. Saxena and S. Rawat
Antibodies
B cells
Stimulatory secretions
Helper T cell
Antigen from a food vaccine Taken up by M cells
M cells passes the antigen to macrophages and B cells
Macrophages display pieces of the antigen to helper T cells
Activated B cells make and release antibodies able to neutralize the antigen
T cells stimulate B cells and seek out antigen to distant site
Memory B cell
cell infection
Infected cell
Arrival of virus/ antigen
Memory helper T cell
Cytotoxic T cell
Memory helper T cells produce cytotoxic T cells to attack infected cells
Memory helper T cells swiftly stimulate antibody
ti
Antibodies quickly neutralize the invader
Initial response
When a disease agent appears
(a)
(b)
Fig. 12.3 a Production ofantibodies. b Neutralizationof invader
12 Edible Vaccines 221
12.9.3 Rabies
Rabies is a deadly viral infection that is trans-mitted to humans from animals. Tomato plantsexpressing rabies antigens could induce anti-bodies in mice. Alternatively, TMV may also beused. Transformed tomato plants using CaMVwith the glycoprotein (G-protein) gene of rabiesvirus (ERA strain) was shown to be immuno-genic in animals.
12.9.4 Hepatitis B
Hepatitis B is a potentially life-threatening liverinfection caused by the hepatitis B virus. It isestimated to have infected 400 million peoplethroughout the globe, making the virus one ofthe most common human pathogen. First humantrials of a potato-based vaccine against hepatitisB have reported encouraging results. Sinceimmunization is the only known method to
Table 12.3 Therapeutic and diagnostic application of edible vaccines
Name of the vaccine Vector Pathological condition
Rabies virus Tobacco, spinach Rabies
Hepatitis B Potato, Tobacco, Banana Hepatitis B
HIV Tomato AIDS
Vibrio cholerae Potato Cholera
Cancer Wheat, Rice Cancer
Norwalk virus Tobacco, potato Hepatitis B
Rabbit hemorrhagic disease virus Potato Hemorrhage
Transmissible gastroenteritis corona virus Tobacco Gastroenteritis
Alzheimer’s disease Tomato Alzheimer’s disease
Colon cancer Tobacco and potato Colon cancer
Paramyxovirus Banana, rice, lettuce Measles
Plasmodium falciparum Tobacco Malaria
Type-I Diabetes Potato Type-I diabetes
Cysticercosis Arabidopsis Cysticercosis, foot and mouth disease
Table 12.2 Details of antigens produced by host plants and antibodies produced against them
Antibody Antigen Plant
IgG Transition stage analog Tobacco
IgM NP(4-hydroxy-3-nitrophenyl) acetyl hapten Tobacco
Single domain (dAb) Substance B Tobacco
Single chain Fv Phytochrome Tobacco
Single chain Fv Artichoke mottled virus coat protein Tobacco
Fab, IgG Human creatine kinase Arabidopsis
IgG Fungal cutinase Tobacco
IgG(k) and SIgG/A hybrid S. mutagens adhesion Tobacco
Single chain Fv Abscisic acid Tobacco
Single chain Fv Nematode antigen Tobacco
Single chain Fv Alpha-glucuronidase Tobacco
IgG Glycoprotein B of herpes simplex virus Soybean
(Das 2009)
222 J. Saxena and S. Rawat
prevent the disease of the hepatitis B virus, anyattempt to reduce its infection requires theavailability of large quantities of vaccineHBsAg. The amount of HBsAg needed for onedose could be achieved in a single potato. Levelsof specific antibodies significantly exceeded theprotective level of 10 mIU/mL in humans. Whencloned into CaMV, the pCMV-S plasmidencoding the HBsAg subtype ayw showedhigher expression in roots as compared to leaftissue of the transgenic potato. Furthermore,expression of the antigen was found to be higherin roots of transgenic potato than in leaf tissues.However, the expression of HBsAg in transgenicpotatoes is not sufficient for using as oral vac-cine. Further studies are underway to increasethe level of HBsAg by using different promoterse.g., patatin promoter, and different transcriptionregulating elements.
12.9.5 Cholera
Cholera is an infection of the small intestine thatcauses a large amount of watery diarrhea. Itcauses up to 10 million deaths per year in thedeveloping world, primarily among children.Studies supported by WHO have demonstratedpossibility of an effective vaccine for cholera,which provides cross protection against entero-toxic E. coli. To address this limitation, plantswere transformed with the gene encoding Bsubunit of the E. coli heat liable enterotoxin(LT-B). Transgenic potatoes expressing LT-Bwere found to induce both serum and secretoryantibodies when fed to mice; these antibodieswere protective in bacterial toxin assay in vitro.This is the first ‘‘proof of concept’’ for the ediblevaccine.
Since people eat only cooked potatoes, theeffect of boiling on the properties of CTBexpressed in transgenic potatoes was examined.After boiling for 5 min, over half of the vaccineprotein survived in its biologically active form,providing evidence that cooking does not alwaysinactivate edible vaccines. Thus, the spectrum of
plants for producing edible vaccines may beexpanded beyond raw food plants such as fruits.Co-expression of mutant cholera toxin subunit(mCT-A) and LT-B in crop seeds has beenshown to be effective by nasal administrationand is extremely practical.
12.9.6 Diabetes
The prevalence of diabetes is increasing globallyand India is no exception. More than 100 mil-lion people are affected with diabetes world-wide. Type-I diabetes, also known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, primarily affects children andyoung adults and accounts for 5–10 % of thediagnosed diabetes in North America. Researchby Ma and Hein (1995) at the University ofWestern Ontario showed that diabetes can beprevented in mice by feeding them with plantsengineered to produce a diabetes related-protein.The idea is based on ‘oral tolerance’ where theautoimmune system is selectively turned offearly by teaching the body to tolerate the‘‘antigenic proteins’’. The pancreatic protein,glutamic acid decarboxylase (GAD67) is linkedto the onset of IDDM, and when injected intomice it is known to prevent diabetes.
The Canadian group developed transgenicpotato and tobacco plants with the gene forGAD67, fed them to nonobese diabetic mice,which developed insulin-dependent diabetesspontaneously. The results were intriguing, only20 % of the prediabetic mice fed with transgenicplants developed the diabetes, while 70 % non-treated mice developed the disease. The treatedmice also showed increased levels of IG1, anantibody associated with cytokines, which sup-presses harmful immune responses. Thus, theantigen produced in plants appears to retainimmunogenicity and prevent diabetes in ananimal model. According to Canadian scientists,this is the first proof of principle for the use ofedible vaccines in the treatment of the autoim-mune diseases.
12 Edible Vaccines 223
12.9.7 HIV
Human immunodeficiency virus (HIV) is a ret-roviral that causes acquired immunodeficiencysyndrome (AIDS), a condition in humans inwhich progressive failure of the immune systemallows life-threatening opportunistic infectionsand cancer to thrive. In order to produce ediblevaccine initial success in splicing HIV proteininto CPMV has been achieved. Two HIV proteingenes and CaMV as promoter were successfullyinjected into tomatoes with a needle, and theexpressed protein was demonstrable by poly-merase chain reaction (PCR) in different parts ofthe plant, including the ripe fruit, as well as inthe second generation plants. Recently, spinachhas been successfully inoculated for Tat proteinexpression cloned into TMV. Each gram of leaftissue of spinach was shown to contain up to300–500 lg of Tat antigen. Mice fed with thisspinach followed by DNA vaccinations resultedin higher antibody titers than the controls, withthe levels peaking at 4 weeks post-vaccination.
12.10 Regulatory Issues
It is still unclear whether the edible vaccineswould be regulated under food, drugs. or agri-cultural products and what vaccine componentwould be licensed—antigen itself, geneticallyengineered fruit or transgenic seeds. They wouldbe subjected to a very close scrutiny by theregulatory bodies in order to ensure that theynever enter the food supply. This would includegreenhouse segregation of medicinal plants fromfood crops to prevent outcrossing and wouldnecessitate separate storage and processingfacilities. Although edible vaccines fall under‘‘Genetically modified’’ plants, it is hoped thatthese vaccines will avoid serious controversy,because they are intended to save lives.
12.10.1 Clinical Trials
Edible vaccines are future vaccines and somechallenges are yet to be overcome before thesecan become a reality. Like all products regulated
by Food and Drug Administration, edible vac-cines undergo a rigorous review of laboratory, andclinical testing that are conducted to get infor-mation regarding safety, efficacy, purity, andpotency of these products. These trials can takeplace only after satisfactory information has beencollected on the quality of the nonclinical safety.
Successful expression of antigens in plantshas been demonstrated in the past. The vaccineshave also been checked for their efficacy inhumans. Results from the primary phase of thefirst-ever human clinical trial of an edible vac-cine were published in the journal NatureMedicine in 1998 (Blaine P. Friedlander, BoyceThomson Institute of Plant Research), whichindicated that consumption of servings of rawpotatoes resulted in immunity to specific dis-eases. The human clinical study was conductedunder the direction of Dr. Carol Tacket at theCenter for Vaccine Development, University ofMaryland School of Medicine in Baltimore. Inthe first phase of human testing, the potatoeseaten by volunteers contained a vaccine againsttravelers’ diarrhea, a common condition result-ing from intestinal infection by the bacteriumE. coli, which contaminates food or water sup-plies. The clinical trials were approved inadvance by the Food and Drug Administration.
Encouraged by the results of this study, sci-entists started exploring the use of this techniquefor administering other antigens. In 2005 Than-avala’s group has developed a potato vaccinebooster for use in conjunction with injectedhepatitis B vaccine. It is currently in phase IIclinical trial and phase I for patients who havepreviously been vaccinated. In 2000, Tacket andhis team mates studied the human immuneresponse to the Norwalk virus capsid proteinexpressed in potatoes. Overall, 95 % (19 out of20 volunteers) developed some kind of immuneresponse, although the antibody increase in somecases was modest. In same year, Pogrebnyak’slab developed an effective vaccine against thecoronavirus which causes severe acute respira-tory syndrome (SARS). Tomato and Tobaccoplants are used for high expression of the coro-navirus spike protein (S1). First, lyophilizedtomato fruit was fed to mice and then boosting
224 J. Saxena and S. Rawat
occurred with S1 protein expressed in tobaccoroots; high IgG1 immune responses and signifi-cant IgG2a and IgG2b responses were observedin their sera. Research is also going in thedirection to engineer the plants to produce avariety of functional monoclonal antibody (Maet al. 2005).
In the first human study of transgenic plantvaccine designed to induce active immunity, 14adult volunteers were given either 100 g of trans-genic potato, 50 g of transgenic potato or 50 g ofwild type potato, each transgenic potatoes con-taining from 3.7 to 15.7 lg/g of LT-B. The variabledose per gram of potato was due to the tissuespecificity of the promoter, therefore, that LT-Bwas expressed to a different degree in the differenttissues of the potatoes. The potatoes in this studywere ingested raw; however, subsequent studieshave shown that transgenic potatoes expressing theB subunit of cholera toxin could be boiled for3 min until the tissue becomes soft with loss ofonly about 50 % of the CT- B pentameric GM1-binding form. Serologic responses were alsodetected after vaccination. Totally 10 out of the 11volunteers (91 %) who ingested transgenic pota-toes developed IgG anti-LT and in half of themresponses occurred after the first dose. There are 6of the 11 (55 %) volunteers developed fourfold risein serum IgA anti-LT.
Researchers supported by the NIAID haveshown for the first time that an edible vaccinecan safely trigger significant immune responsesin people. The goal of the Phase 1 proof-of-concept trial study was to demonstrate that anedible vaccine could stimulate an immuneresponse in humans. Volunteers ate bite-sizedpieces of raw potato that had been geneticallyengineered to produce part of the toxin secretedby E. coli, which causes diarrhea. The trialenrolled 14 healthy adults, 11 were chosen atrandom to receive the genetically engineeredpotatoes and 3 received pieces of ordinarypotatoes. The investigator periodically collectedblood and stool samples from the volunteers toevaluate the vaccine’s ability to stimulate bothsystemic and intestinal immune responses. Thepotatoes were well tolerated and no one expe-rienced serious adverse side effects.
NIAID supported scientists are exploring theuse of this technique for administering otherantigens. Edible vaccines for other intestinalpathogens are already in the pipeline. Potatoesand bananas that might protect against Norwalkvirus, a common cause of diarrhea, and potatoesand tomatoes that might protect against hepatitisB are being developed.
12.11 Future Perspectiveand Conclusion
Thirty million children throughout the world donot receive even the most basic immunizationseach year. As a result, at least three million ofthese children die from diseases that are fullyvaccine-preventable. The solution to vaccinatethese children might seem simple with the ideaof large scale production of edible vaccines forvarious diseases.
As a recent progress, the first human clinicaltrials for plant-based vaccine have been per-formed; it brings many challenges like optimi-zation of expression levels, stabilization duringpost harvest storage, etc. Long-term reactions toedible vaccines are yet to be determined. Pos-sible delayed reactions not yet discovered maybe the point of consideration. In addition to that,edible vaccines can be further improved for theiroral immunogenicity by the use of specificadjuvant which can be applied either as a fusionto the candidate gene or as an independent gene.Some of the diseases to which edible vaccineshave shown promising application may beelaborated in the veterinary as well as humanspectrum. These studies conclude plant-derivedvaccines as a new hope and promise for moreimmunogenic, more effective, and less expen-sive vaccination strategies against both respira-tory as well as intestinal mucosal pathogens.
Research in the field of edible vaccines holdsimmense potential for the future and everyadvancement made in this direction is bringingthe dream of edible vaccine one step closer.There is hope that in coming future edible vac-cines will conquer all serious diseases and makethe planet beautiful to live in.
12 Edible Vaccines 225
References
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Chowdhury K, Bagasra O (2007) An edible vaccine formalaria using transgenic tomatoes of varying sizes,shapes and colors to carry different antigens. MedHypo 68:22–30
Das DK (2009) Plant derived edible vaccines. Curr TrenBiotech Pharm 3(2):113–127
Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU(1999) In vivo analysis of plastid psbA, rbcL andrpl32 UTR elements by chloroplast transformation:tobacco plastid gene expression is controlled bymodulation of transcript levels and translation effi-ciency. Plant J 19:333–345
Gruissem W, Tonkyn J (1993) Control mechanisms ofplastid gene expression. CRC Critical Rev Plant Biol12:19–55
Hiatt A, Cafferkey R, Bowdish K (1989) Production ofantibodies in transgenic plants. Nature342(6245):76–78
Ma JKC, Hein MB (1995) Immunotherapeutic potentialof antibodies produced in plants. Trends Biotechnol-ogy 13:522–527
Ma S, Huang Y, Davis A, Yin Z, Mi Q, Menassa R,Brandle JE, Jevnikar AM (2005) Production ofbiologically active human interleukin-4 in transgenictomato and potato. Plant Biotechnol J 3(3):309–318
Mason HS, Arntzen CJ (1995) Transgenic plants asvaccine production systems. Trends Biotechnol13:388–392
Mason HS, Lam DMK, Arntzen CJ (1992) Expression ofhepatitis B surface antigen in transgenic plants. ProcNatl Acad Sci USA 89:11745–11749
Mason HS, Haq TA, Clements JD, Arntzen CJ (1998)Edible vaccine protects mice against Escherichia coliheat-labile enterotoxin (LT): potatoes expressing asynthetic LT-B gene. Vaccine 16:1336–1343
Mason HS, Warzecha H, Tsafir MS, Arntzen CJ (2002)Edible plant vaccines: applications for prophylacticand therapeutic molecular medicine. Trends Mol Med8:324–329
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Richter LJ, Thanavala Y, Arntzen CJ, Mason HS (2000)Production of hepatitis B surface antigen in trans-genic plants for oral immunization. Nat Biotechnol18(11):1167–1171
Tacket CO, Mason HS, Losonsky G, Estes MK, LevineMM, Arntzen CJ (2000) Human immune responses toa novel Norwalk virus vaccine delivered in transgenicpotatoes. J Infect Dis 182:302–305
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226 J. Saxena and S. Rawat
13Engineering Plantsfor Phytoremediation
Mamta Baunthiyal
Abstract
In order to restore environmental balance, the utility of phytoremediationto remediate environmental contamination has received much attention inthe last few years. Considerable effort has been devoted to making thetransition from the laboratory to commercialization. Although plantshave the inherent ability to detoxify contaminants, they generally lack thecatabolic pathway for the complete degradation of these compounds ascompared to microorganisms. There are also concerns over the potentialfor the introduction of contaminants into the food chain and how todispose of plants that accumulate them in high quantities is also a seriousconcern. Hence, the utility of phytoremediation to remediate environ-mental contamination is still somewhat in question. For these reasons,researchers have endeavored to engineer plants with genes that canbestow superior degradation abilities. Genes from microbes, plants, andanimals are being used successfully to enhance the ability of plants totolerate, remove, and degrade pollutants. Although improvement ofplants by genetic engineering opens up new possibilities for phytoreme-diation, it is still in its research and development phase, with manytechnical issues needing to be addressed.
13.1 Introduction
Elemental pollutants such as arsenic (As), cop-per (Cu), cadmium (Cd), mercury (Hg), zinc(Zn), and lead (Pb) are difficult to remediatefrom air, water, and soil because these toxic
elements are immutable by all biochemicalreactions, hence remain in the ecosystem. Theyseep into surface water, groundwater, or soil andmay channel into the food chain through cropsgrowing on such soil and water. Apart fromthese metals, small organic contaminants andvolatile organic compounds are other prevalentcontaminants of the environment. These includepetroleum hydrocarbons (PHC), halogenatedhydrocarbons, polycyclic aromatic hydrocarbons(PAHs), polychlorinated biphenyls (PCBs) andpesticides, and solvents like trichloroethylene(TCE), carbon tetrachloride, and salts. PAHs, in
M. Baunthiyal (&)Govind Ballabh Pant Engineering College,Ghurdauri, Pauri, Uttarakhand 246194, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_13, � Springer India 2014
227
general, are ubiquitous environmental pollutantsand are formed from both natural and anthro-pogenic sources. Anthropogenic sources are byfar the major contributors and include theburning of coal refuse banks, coke production,automobiles, commercial incinerators, and woodgasifers. PAHs are an environmental concernbecause they are not only toxic to aquatic lifebut also several are suspected humancarcinogens
The term phytoremediation (‘‘phyto’’ mean-ing plant, and the Latin suffix ‘‘remedium’’meaning to restore) refers to a diverse collectionof plant-based technologies that use plants(either naturally occurring or genetically engi-neered) to clean contaminated environments.Phytoremediation is popular because of its cost-effectiveness, environmentally friendly aestheticadvantages, long-term applicability, and goodinfluence on the physical, chemical, and bio-logical aspects of the environment. The tech-nology can be used at hazardous waste siteswhere other methods of treatment are tooexpensive or impractical; it may also be used atlow-level contaminated sites where only ‘‘pol-ishing treatment’’ is required over a long periodof time. Compared to the use of other organisms,the use of plants costs less as they capture solarenergy to synthesize the proteins and otherstructures that are necessary for the remediation.Phytoremediation can be used in conjunctionwith other technologies as a final cap. Phytoex-traction, a type of phytoremediation, is aneffective means of remediating a site because itreduces the overall mass to be treated from tonsof widespread contaminated soil to plant tissuethat can be dried to a small volume. Unlike otherengineering methods that might remove thefertile topsoil, phytoremediation does not reducethe fertility of the site (Chaney et al. 1997).
To be effective, the concentration of themetal in the harvestable part of the plants mustbe higher than its concentration in the soil sothat the volume of the hazardous plant waste isless than the volume of the contaminated soil.Plants that are especially good at concentratingthe pollutants are termed as hyperaccumulators.The ‘bioconcentration factor’ (BCF) refers to the
metal concentration in plant tissues relative tothe metal concentration in the substrate, and avalue [1 indicates that the plant actively con-centrates the metal. A metal hyperaccumulator isdefined as a plant that can concentrate the metalsto a level of 0.1 % for nickel (Ni), cobalt (Co),Cu, and Pb, 1 % for Zn and 0.01 % for Cd(Baker et al. 2000). For example, Pteris vittata(Chinese brake fern) efficiently hyperaccumu-lates arsenic in its fronds. Another hyperaccu-mulator is Thlaspi caerulescens, which canconcentrate cadmium, a highly toxic and prob-ably carcinogenic metal, in the above-groundtissues at concentrations 1,000 times higher thanthe normal toxic concentration of only 1 ppm.
Although plants have innate capabilities ofremedying hazardous contaminants from theenvironment, the rate is directly proportional toplant growth rate and the total amount ofremediation is correlated with the plant totalbiomass, making the process very slow. Further,phytoremediation using natural plants has otherlimitations that include the potential for intro-ducing the contaminant or its metabolites intothe food chain, long cleanup times required toachieve regulatory action levels, and toxicityencountered in establishing and maintainingvegetation at waste sites. The plants may alsosuffer toxicity symptoms.
Keeping these limitations in view, it would bebeneficial to enhance plants’ capacity for phy-toremediation. This necessitates the identifica-tion of a fast growing (largest potential biomassand greatest nutrient responses) and morestrongly metal accumulating genotype. Geneticengineering provides an excellent means toachieve this goal. It is possible to generatetransgenic plants that can overexpress theirgenes or express exogenous genes, and therebyacquire new or improved phenotypes. One majoradvantage of genetically engineered plants isthat the ability of specific enzymes for degra-dation of a contaminant could be transferred to aplant species that is either indigenous to anecosystem or has other desirable remediationproperties such as rapid growth, deep rootstructures, or high water uptake. Once anindividual plant with suitable phenotype is
228 M. Baunthiyal
produced, it is easy to multiply it by cloningmethods, such as cuttings and somatic embryo-genesis. Moreover, many trees continue to growfor many years as long as the conditions aresuitable.
13.2 Genetic Engineering Strategiesfor Plant Modificationto Enhance Phytoremediation
Many transgenic plants with new characteristicsdue to expression of genes from microorgan-isms, yeasts, mouse, or even human have beenreported in scientific journals. Till date, allcommercial and research activities have usednaturally occurring plant species. Many of theseare species that can be genetically engineered. Ingeneral, any dicotyledonous plant species can begenetically engineered using the Agrobacteriumvector system, while most monocotyledonousplants can be transformed using particle gun orelectroporation techniques.
Recent studies using transgenic aspen over-expressed with a nitroreductase, pseudomonasnitroreductase A (pnrA), isolated from the bac-terium Pseudomonas putida have shown higheraccumulation of TNT (2, 4, 6-trinitrotoluene)from liquid culture and soil, compared to
nontransgenic plants. Furthermore, the tolerancelimit toward TNT was also significantly higherthan for nontransgenic plants.
The genetic engineering approach has suc-cessfully facilitated to alter the biological func-tions of plants through modification of primaryand secondary metabolism and by adding newphenotypic and genotypic characters to plantswith the aim of understanding and improvingtheir phytoremediation properties (Fig. 13.1).Transgenic plants produced for metabolizingherbicides and long-persisting pollutants can beused for phytoremediation of foreign chemicalsin contaminated soil and water (Dowling andDoty 2009).
Metal-hyperaccumulating plants andmicrobes with unique abilities to tolerate,accumulate, and detoxify metals and metalloidsrepresent an important reservoir of unique genes.These genes could be transferred to fast-growingplant species to improve phytoremediation(Fulekar et al. 2009). It has been established thatthe adaptive metal tolerance is governed by asmall number of major genes and some minormodifier genes. Probably, it is this adaptivemetal tolerance that gears a plant species forhyperaccumulation.
Use of tissue culture techniques to selectgenes having enhanced bio-degradative proper-ties (for organics) or the enhanced ability to
Fig. 13.1 Geneticengineering inphytoremediation
13 Engineering Plants for Phytoremediation 229
assimilate metals helps to select plants with thedesired characters. Molecular techniques such asthe analysis of molecular variance of the randomamplified polymorphic DNA (RAPD) markersare useful to investigate the genetic diversity andheavy metal tolerance in plant populations,providing the opportunity to investigate the firststeps in the differentiation of plant populationsunder severe selection pressure and to selectplants for phytoremediation.
The genetic and biochemical basis isbecoming an interesting target for genetic engi-neering. A fundamental understanding of bothuptake and translocation processes in normalplants and metal hyperaccumulators, regulatorycontrol of these activities, and the use of tissuespecific promoters offers the ability to developeffective and economic phytoremediation plantsfor soil metals. Examples include genes con-trolling the synthesis of peptides that sequestermetals, like phytochelatins, genes encodingtransport proteins, or genes encoding enzymesthat change the oxidation state of heavy metals.The genes involved in the metabolism ofchemical compounds can be isolated from vari-ous organisms, including bacteria, fungi, plants,and animals, and these genes are then introducedinto candidate plants. The desired characters forphytoremediation can be improved by identify-ing candidate protein, metal chelators, andtransporter genes for transfer and/or overex-pression of a particular gene.
13.2.1 Plant Metal Transporters
One of the promising approaches to enhance theability of metal ions to enter plant cells is theidentification of the metal transporter proteinsand introducing genes encoding transportermolecules. These are generally proteins found inthe cell membrane, that either have an affinityfor metal ions, or that create favorable energeticconditions to allow metals to enter the cell. Tilldate, several plant metal transporters have been
reported and more remain to be recognized.Some of the transporters identified so far includethe Arabidopsis IRT1 gene that encodes a pro-tein that regulates the uptake of iron and othermetals (Eide et al. 1996) and the MRP1 geneencoding, an Mg-ATPase transporter, also fromArabidopsis (Lu et al. 1997).
Further, success in this approach is achievedby identifying proteins such as ZNT1, ZIP1-4,IRT1, COPT1, LCT1, and tVramp-1/3/4 on theplasma membrane-cytosol interface; ZAT, ABCtype, HMT1, AtMRP, CAX2 seen in vacuoles;and RAN1 in Golgi bodies. Manipulations ofthese transporters to achieve the removal ofmetal ions from the cell hold great potential(Tong et al. 2004). The natural resistance-associated macrophage proteins (Nramp) familyof transporters has been recently characterizedfrom rice and Arabidopsis.
13.2.2 Phytochelatins for MetalSequestering
The principal classes of metal chelators includephytochelatins, metallothioneins, organic acids,and amino acids. Phytochelatins (PCs) are smallmetal-binding peptides found in plants. Iso-PCs,a series of PC-like homologous chelating pep-tides, are reported with varying terminal aminoacids and have a C-terminal modified residueother than glycine. The PC and iso-PC mole-cules form complexes with heavy metals likeCd. In addition to PC-Cd complex other PC-metal complexes include Ag, Cu, and As (Shahand Nongkynrih 2007).
In vitro experiments have shown that a seriesof metal-sensitive plant enzymes can tolerate a10- to 1,000-fold concentration of Cd in theform of a PC complex than as free radical ion.PC reactivate metal poisoned plant enzymessuch as nitrate reductase up to 1,000-fold betterthan chelators such as glutathione (GSH) orcitrate, signifying the extraordinary sequesteringpotential of these peptides.
230 M. Baunthiyal
13.2.3 Metallothioneins
Metallothioneins (MTs) are metal-binding pro-teins that confer heavy metal tolerance andaccumulation (Hamer 1986; Malin and Bülow2001). These are low molecular mass cysteinerich proteins that were originally isolated as Cu,Cd, and Zn binding proteins in mammals. MTsare able to bind a variety of metals by the for-mation of mercaptide bonds between thenumerous cysteine (Cys) residues present in theproteins and the metal, and it is the arrangementof these residues that in part determines themetal-binding properties of the MT proteins.Researchers successfully reported more than50 MTs in different plants categorized into fourtypes, types 1–4. These are based on the Cysarrangement. Although MTs are expressedthroughout the plant, some have been found tobe expressed in a tissue-specific manner.Transgenic plants expressing MTs have beencreated, and although these plants exhibitedenhanced tolerance to high metal concentrations,the uptake of metals was not enhanced. Toenhance higher plant metal sequestration, theyeast MT CUP1 was introduced into tobaccoplants, and the cup 1 gene expression and Cuand Cd phytoextraction were determined.Overexpression of Cu inducible MT cup 1 alsoenhanced Cu tolerance in plants. In plants, awide range of MT genes from various sourceshave been overexpressed including those fromhuman, mouse, Chinese hamster, and yeast.
The vacuole is generally considered to be themain storage site for metals in yeast and plantcells and there is evidence that phytochelatin-metal complexes are pumped into the vacuole.The best characterized of the known vacuolartransporters and channels involved in metaltolerance are YCF1 from yeast, Saccharomycescerevisiae. YCF1 is an MgATP-energized glu-tathione S-conjugate transporter responsible forvacuolar sequestration of compounds aftertheir S-conjugation with glutathione. Overex-pression of the YCF1 gene in Arbidobsis thali-ana and the YCF1 proteins were found to beassociated with the tonoplast and the plasma
membrane. The vacuoles of the YCF1-transgenicplants exhibited a 4-fold higher rate ofglutathione-Cd uptake than those of wild-typeplants, indicating that expression of YCF1 -strongly increases Cd transport activity. Thetransgenic plants showed improved resistance toboth Cd and Pb and elevated metal content,characteristics desirable for phytoremediation.
13.2.4 Genes to Change the OxidationState of Heavy Metals
Genes may be introduced that code for enzymesto change the oxidation state of heavy metalslike Hg and selenium (Se). For instance, intro-duction of the bacterial merA gene encodingmercuric oxide reductase (Rugh et al. 1996), orthat which converts metals into less toxic forms,such as enzymes that can methylate Se intodimethylselenate (Hansen et al. 1998). In boththese cases, the resulting form of the metal isvolatile, so that one can create a plant capable ofmetal remediation by phytovolatilization,another type of phytoremediation.
13.3 Genetically Modified Plantsfor Metal Uptake, Tolerance,and Detoxification
Several researchers have already reportedencouraging results using plants bioengineeredwith increased heavy metal tolerance and uptakeof heavy metals for the purpose of phytoreme-diation. The majority of these novel plants haveonly been tested under limited laboratory con-ditions and very few have been grown in thefield (Table 13.1).
13.3.1 Arabidopsis
Arsenic is an extremely toxic metalloid pollutantand the decontamination of polluted sites can beenvironmentally destructive. It is a lethal poisonthat is released into the environment from
13 Engineering Plants for Phytoremediation 231
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232 M. Baunthiyal
natural processes and via arsenic-based chemi-cals. Genetically engineered Arabidopsis plantscan sequester As from the soil. These plants cantransport As aboveground, reduce it to arsenite,and sequester it in thiol-peptide com-plexes (Dhankher et al. 2002). By coexpressingtwo bacterial genes, arsenate reductase (ArsC)and c-glutamylcysteine synthetase (c-ECS) inArabidopsis plants, it was observed that plantsexpressing SRS1p/ArsC and ACT2p/c-ECStogether showed substantially greater As toler-ance than wild-type plants or plants expressingc-ECS alone. In addition, when grown on As,these plants accumulated 4- to 17-fold greaterfresh shoot weight and accumulated 2- to 3-foldmore As per gram of tissue than wild-type plantsor plants expressing c-ECS or ArsC alone.
Extensive progress has also been achieved inidentifying genes and proteins involved inuptake of iron (Fe) by yeast and plants. Asdescribed earlier, the utility of the yeast proteinYCF1, a protein which detoxifies Cd by trans-porting it into vacuoles, has been implementedfor the remediation of Cd and Pb. Transgenic A.thaliana plants overexpressing YCF1 showed anenhanced tolerance and accumulated greateramounts of Cd and Pb.
The close relationship between Arabidopsishalleri, a metal tolerant and hyperaccumulatingrelative of the biological model species A. tha-liana, has recently allowed the use of A. thalianaGeneChips to compare gene expression levelsbetween A. halleri and the nontolerant A. thali-ana and, consequently, permitted the identifica-tion of genes potentially involved in metaltolerance and/or hyperaccumulation. The com-plete annotation of the A. thaliana genomesequence provides a solid foundation for com-parative mapping studies within the Brassicaceaefamily. The genome organization of A. thalianahas already been compared with those of severalspecies like Arabidopsis lyrata, Arabidopsispetraea and Capsella rubella. Moreover, A.halleri is a species that has undergone naturalselection for Zn tolerance. Isolation of thequantitative trait loci (QTL) associated with thistrait holds great promise for the identification ofthe main genes responsible for this adaptation.
13.3.2 Brassica
Brassica juncea was genetically engineered toinvestigate rate-limiting factors for glutathioneand phytochelatin production. To achieve thisEscherichia coli gshII gene was introduced.Cd-treated GS plants had higher concentrations ofglutathione, phytochelatin, thiol, sulphur, andcalcium than wild-type plants (Zhu et al. 1999). Astudy showed that c-glutamylcysteine synthetaseinhibitor, L-buthionine-[S,R]-sulphoximine (BSO),dramatically increased As sensitivity both innonadapted and As-hypertolerant plants, showingthat phytochelatin-based sequestration is essen-tial for both normal constitutive tolerance andadaptative hypertolerance to this metalloid (Schatet al. 2002).
Astragalus bisulcatus is a native plant thathas the capacity to grow on Se containing soilsand accumulate Se to high concentrations but ithas a slow growth rate. It has been proposed thatin A. bisulcatus selenocysteine methyltransfer-ase (SMT) converts the amino acid SeCys intothe nonprotein amino acid (MetSeCys). Byincorporating gene for SMT from the Sehyperaccumulator A. bisulcatus, it diverted theflow of Se from the Se amino acids that mayotherwise be incorporated into protein, leadingto alterations in enzyme structure, function, andtoxicity. Transgenic plants overexpressing SMTshow enhanced tolerance to Se, particularlyselenite, and produced 3- to 7-fold more biomassthan wild type and 3-fold longer root lengths(LeDuc et al. 2004). Indian mustard plantsoverexpressing the A. bisulcatus smt gene,exhibited a greatly increased accumulation ofMetSeCys and tolerance to Se compounds, inparticular selenite.
13.3.3 Tobacco
Scientists at the University of Cambridgeexpressed the bacterial gene encoding pentae-rythritol tetranitrate reductase in transgenictobacco, conferring the ability to survive ongrowth media containing otherwise toxic levelsof the nitrate ester class of explosives. Further
13 Engineering Plants for Phytoremediation 233
analysis also demonstrated an enhanced degra-dation of these compounds by transgenic tobaccoplants relative to untransformed seedlings.
13.3.4 Tomato
When bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase was expressedin tomato plants, it showed enhanced metalaccumulation and tolerance levels for a range ofheavy metals (Cd, Cu, Ni, Mg, Pb, and Zn) thanuntransformed plants (Grichko et al. 2000).
13.3.5 Pteris vittata
Pteris vittata can effectively remove As metal-loid from soil. For example, in soil contaminatedwith As at a concentration of 97 ppm, the olderfronds of the fern had As concentrations of up to3894 lg per gram of tissue. Less than 168 lg Asper gram was found in the root tissue. More than95 % of the As removed from the soil by thefern was translocated to the aboveground bio-mass (Doty 2008). Unfortunately, this plantspecies grows well only in warm, humidenvironments.
13.3.6 Poplar and Willow
Although the research on several hyperaccu-mulating species is promising, the speciesthemselves are too small and slow-growing forsome phytoremediation applications. For thisreason, high-biomass crops such as poplar andwillow are being studied for phytoremediationof metals. Poplar and willow are not hyperac-cumulators as they do not concentrate metals tohigh concentrations, but because of their greaterbiomass and deep root systems, they are alsoeffective remediators of metal contamination.Poplar plant transformation was conducted usingAgrobacterium-mediated transfer of genes.Transgenic poplar plantlets which express genesof interest were then multiplied by cuttings.
After multiple-year field trials, it can be con-firmed whether transgenic poplar plants areindeed improved in the capacity to remediate thepolluted environment, thus suitable for actualphytoremediation. There is a possibility thatplants with single gene insertion may not besufficiently improved in the capacity to remedi-ate the actual field. To make the process moreeffective, multiple genes with different andsynergistic mechanisms of phytoremediation areexpressed at the same time. To develop moreand better plants for phytoremediation, thesearch for new genes with different functionsand stacking them (introducing many genes intothe same line of plants) will hopefully furtherimprove the plants’ capacity to clean up theenvironment.
Willow was specifically suggested for phy-toremediation of heavy-metal contaminatedlands because of its unique ability to re-growreadily after its shoots have been harvested, adistinctive trait of willow. Further there is sub-stantial genetic diversity of willow, with over450 Salix species. Willow plants (Salix matsu-dana 9 Salix alba NZ1040) grown in soil con-taminated with Cd at concentrations commonlyfound in agricultural sites fertilized with Cd-containing fertilizers accumulated Cd in theaboveground tissues with BCFs of about 10(Doty 2008).
13.4 Challenges
To improve the process of phytoremediationusing genetic modifications success has beenachieved, and these processes have greatpotential for field applications, the only con-straint being public acceptance of geneticallymodified organisms.
The key concerns in remediating contami-nated sites by transgenic plants include:1. Characterization of vector system, its stabil-
ity, and expression in the plant cell.2. Avoidance of unwanted properties encoded
by introduced DNA like toxic, pathogenic, ordeleterious functions.
234 M. Baunthiyal
3. Potential risks to birds and insects that mightfeed on these plants.
4. Proper disposal of plant biomass containinghigh concentrations of hazardous substances,particularly metals.
5. Competitiveness of transgenic to wild type.6. The ability to outcross with related species
(particularly wild relatives).
13.4.1 Complexity of Phytoremediationin the Field
Uneven distribution of contaminants is the fre-quently encountered challenge in field studies,including ‘‘hot spots’’ across a site. In labora-tory and greenhouse experiments, soils generallyhave uniform matrix. This may not be possiblein the field even if the site is extensively tilledprior to planting; this spatial heterogeneity ofinitial contaminant levels results in data scatter,which can make it difficult to statistically showsignificant treatment effects for field trials. Thisis also problematic in terms of regulatory stan-dards because remediation success is oftenjudged on a point-by-point basis rather than anaverage of data points from across the site. Eachand every point should meet the regulatory tar-gets; otherwise, the whole site fails.
Other factors such as soil pH, moisture con-tent, root structure, soil structure, organic com-position of the soil, and microbial activity oftenexhibit spatial variability at a given site and canchange over time. One way to mitigate theproblem of spatial variability is to increase thenumber of samples analyzed per treatment plot.However, extensive sampling can lead to highlyvariable data that is difficult to interpret.
Agronomic techniques are often employedprior to planting. Tilling and addition of nutri-ents and organic matter are generally designed toimprove soil texture and composition to facili-tate plant and/or microbial growth. They alsocause changes in soil pH and oxygen content,which can affect the bioavailability, hence deg-radation of contaminants even in bulk (non-rhizosphere) soil. Nutrients in the bulk soil, such
as nitrogen and phosphorus, will positivelyaffect the indigenous microbial populations,including contaminant degraders. Aeration offield soil increases its oxygen concentration,which promotes oxidative degradation ofnumerous organic contaminants. This may alsolead to photo-oxidation of previously buriedorganics. Hence, there is often a decrease incontaminant concentrations in bulk soil (gener-ally used as the negative control) as well as intreatment plots. As a result it is difficult to sta-tistically show that phytoremediation is superiorto natural attenuation, particularly in the initialstages of a field trial. Less remediation isthought to occur in the rhizosphere (2 mm zoneextending from the roots), and much less in bulksoil. Determining the boundary between rhizo-sphere and bulk soil is almost impossible. Fur-thermore, it is impractical to definitivelyseparate fine roots, soil directly in contact withthe roots, and rhizosphere soil into differentsamples for analyses. This can present an ana-lytical challenge for researchers endeavoring toshow PHC and PCB remediation where rhizo-remediation is thought to occur at the root–soilboundary (rhizoplane). There is evidence thatsome plants can provide the impetus for move-ment of hydrophobic organics such as PAHsfrom bulk soil into the rhizosphere (Liste andAlexander 2000). Mobilization of PAHs may beaccelerated by the release of organic acids fromroots, which putatively increases PAH solubilityand bioavailability.
13.4.2 Physical Restrictions and StressFactors
Despite increasing interest in situ remediation ofcontaminants, the number of reports pertainingto phytoremediation appears to be declining,suggesting a decrease in the activity for this areaof research. Numerous unsuccessful and incon-clusive field trial reports provide some insightinto the challenges that researchers haveencountered in this field (Gerhardt et al. 2009).The slower rate of remediation compared to
13 Engineering Plants for Phytoremediation 235
other strategies, such as excavation and ex situtreatment, is a frequently cited disadvantage ofphytoremediation. This may be probablybecause plant growth is hard to achieve inheavily impacted soils, thereby limiting catabo-lism of contaminants. Factors that affect phyto-remediation in the field are not encountered inthe laboratory or greenhouse. These includestressors like plant pathogens; herbivore (insectsand/or animals); variations in temperature,nutrients and precipitation; and competitionfrom weed species that are better adapted to thesite. Any of these abiotic or biotic stressors canslow down or prevent plant growth in the field.For example, attempted phytoremediation ofPHC and other organics at a hydrocarbon burnfacility at NASA Kennedy space center faileddue to drought and competition from weeds(http://www.cluin.org).
There are other physical challenges that limitthe use of phytoremediation. For instance,increasing the moisture content of hydrophobicPHC-contaminated soils can be problematiconce they have become dry, which can preventgermination and seedling growth. Anotherproblem is faced when the contaminated soil isdeeper than the rooting zone, e.g., average rootdepth of herbaceous plants is 50 cm. In thatcase, there is a requirement of excavation priorto phytoremediation. Trees have deeper roots(average 3 m, with much longer roots in somespecies) which can facilitate remediation atgreater depths without excavation. Dendro-remediation (phytoremediation using trees) ofexplosives, e.g., TNT and TCE from soil andgroundwater has shown great promise (VanAken et al. 2004). However, here the challengeis that it can be difficult to grow and establishtrees in contaminated soils. The process is quitelong and they take several years to attain suffi-cient biomass for efficient rates of phytoreme-diation. Further, in case of trees there may be adisposal problem if the roots and wood aredeemed to be contaminated after completion ofremediation.
13.4.3 In-field Performanceof Genetically ModifiedOrganisms
Genetically engineered microbial strains oftenfail to compete with native microbes in the rhi-zosphere, and their numbers diminish to levelsthat merely support remediation.
Degradation of organic contaminants gener-ally requires the concerted action of numerousenzymes and it is generally impractical tointroduce all the genes required for degradationof an organic contaminant into a single plant ormicrobial genome. It can be difficult to stablymaintain even a single gene in transformed orrecombinant organisms and the desired trait,such as enhanced degradative capacity, is oftenlost. For example in plants, silencing of transg-enes, via mechanisms such as cytosine methyl-ation, makes the use of this technologyinherently unpredictable.
Though genetically modified organisms in thelaboratory and greenhouse for phytoremediationhas made considerable progress, regulatoryrestrictions for in situ applications have pre-vented any substantial accumulation of fielddata. There is the potential for inserted geneticmaterial to be transferred to indigenous popu-lations if genetically modified organisms areused without adequate containment systems.Further, recombinant strains that contain antibi-otic resistance genes from the cloning proce-dures cannot be released in the environment.Besides, there has also been low public accep-tance of genetic engineering.
13.4.4 Regulatory Acceptability
The issue of regulatory acceptability for phyto-remediation of hazardous waste is to ensurepublic safety and to protect the environment. TheGovernment regulators must be convinced that agiven remedial strategy will diminish contami-nant toxicity, mobility, and/or concentration
236 M. Baunthiyal
before it is approved for use in the field. Bioac-cumulation of hazardous compounds in plants isone consideration. In situations where plants donot catabolize the contaminants, provisions maybe required for removal of contaminated plantmaterials as part of a remedial plan. Anotherconsideration is the potential ecological risks ofintroducing non-native plant and microbial spe-cies into field sites. These species can move fromthe contaminated site by displacing, or hybrid-izing with native species.
Phytoremediation of organic compoundsoften takes place in the rhizosphere of fine rootsthat have a high turnover rate. These roots havea very short life and the decaying root tissue isconverted into bulk soil during humification.Contributions of organic matter from thedegraded root tissue can complicate sampleanalyses due to the structural similarities tocompounds being remediated. Most of the reg-ulatory agencies for example, United StatesEnvironmental Protection Agency (EPA) andCanadian Council of Ministers of the Environ-ment (CCME), do not distinguish between pe-trogenic and phytogenic carbon compounds.This means that in the case of PHC remediationnaturally occurring organic matter that is co-extracted with organic contaminants can easilylead to exaggerated PHC levels in soil samples.
13.5 Overcoming the Challenges
13.5.1 Chloroplast Engineering
Transformation of chloroplast prevents theescape of transgenes via pollen to related weedsand crops. This method was used to stablyintegrate the bacterial merAB operon into thechloroplast genome of tobacco. The resultingplants were substantially more resistant to highlytoxic organic mercury (Bizily et al. 2000), in theform of phenyl mercuric acetate, than wild type.Other important advantages of chloroplasttransformation include the fact that codon opti-mization is not required to improve expressionof bacterial transgenes.
13.5.2 Minimizing Ecological Risk
To minimize the risks of introducing non-nativebiota (including genetically modified species)into an ecosystem, the best strategy is to usenative species for phytoremediation. It can beapplied to microbial species that are used tofacilitate plant growth and/or degrade contami-nants (Davison 2005). Using native plant speciescan serve the dual purpose of remediation aswell as native habitat reconstruction/reclamation(for microbe-assisted phytoremediation), whichmay be required after successful remediation.
To minimize ecological risks from non-native(transgenic and nontransgenic) phytoremedia-tion species, a biological containment system isoften employed. For example, genes can beintroduced to prevent propagation, or to render aspecies overly sensitive to abiotic stressors suchas temperature changes or chemicals. Multipletransgenes are employed to prevent gene flowbetween the introduced species and other speciesin the environment. This containment systemcan be reinforced by adding mitigator geneslinked to the primary transgene. Mitigatorsgenes confer non-deleterious traits to the phy-toremediation species, but are harmful to relatespecies in case of gene transfer. Alternatively,they can prevent the phytoremediation speciesfrom successfully competing outside the con-taminated site.
13.5.3 Overcoming the Challengeof Plant Stress
Naturally occurring endophytic bacteria havebeen found to degrade organic contaminantssuch as nitro-substituted explosives. In contrastto root-colonizing microbes, endophytes colo-nize internal plant tissues, including root, leaf,and vascular tissues. Inoculating plants withendophytes can thwart competitive pressures inthe rhizosphere as well as some of the challengesthat limit effectiveness of root colonizers,including dependence on specific soil pH, tem-perature, and soil moisture content for optimum
13 Engineering Plants for Phytoremediation 237
growth. Field trials will be required to assess theeffectiveness of the use of endophytes.
13.5.4 Monitoring, Sampling,and Analyzing ExperimentalData from the Field
To demonstrate adequate performance of reme-diation systems in the field, government agen-cies are focusing on issues that address theinconsistencies in experimental protocols, par-ticularly regarding which analytical parametersneed to be measured. Remediation TechnologiesDevelopment Forum, a group with government,industry, and academic partners has developedthe protocol of phytoremediation of PHCs in soilin the 1990s. This forum intends to developprotocols that would allow comparative tests ofremedial strategies at numerous and variedgeographical locations. These protocols recom-mend standards for plot size; number of repli-cations; choice of plant species; plant and soilsampling procedures; microbial and hydrocar-bon analyses; statistical treatment of data; time-points and end-point (3 years). A three-year end-point is considered more realistic than remedi-ation in a single growing season. Establishing alonger time frame for field trials is alwaysadvantageous for researchers because ambigu-ous results are often observed in short-term fieldstudies as a result of tilling and amending thesoil at the onset of a field trial. The three-yearframe duration also allows for assessment of thephytoremediation system and improvement ofmethods between field seasons. If any problemsin the field are encountered, laboratory andgreenhouse experiments can be conducted toresolve the issues prior to the onset of the nextfield season.
One way to facilitate statistical analysis offield data is to use particularly recalcitrantcompounds in the soil samples to normalize thedata. These are called internal markers (alsocalled ‘‘conservative biomarkers’’). They are notgenerally degraded to any appreciable degreeduring phytoremediation. Concentrations ofindividual contaminants can be directly
compared (normalized) to the internal marker.The relative ratio of a specific compound to theinternal marker decreases as remediation occurs.For example, Hopanes, compounds found incrude oil, can be used as biomarkers for PHCremediation, including PAHs.
The methods developed to distinguish bio-logically derived organic compounds fromorganic contaminants can be used for chemicalfingerprinting to assess liability after accidentalrelease of chemicals into the environment. Thesemethods can also be used to distinguish plant-derived hydrocarbons from petrogenic hydro-carbons for assessing phytoremediation.Although these methods are not widelyemployed in the phytoremediation field, in caseof PHC phytoremediation it becomes necessaryto distinguish contamination from naturallyoccurring and anthropogenic hydrocarbons (i.e.,background hydrocarbons). A number ofhydrocarbon indices have been derived to assessthe source of environmental hydrocarbons,including the carbon preference index, averagechain length, and various n-alkane/acyclic iso-prenoid ratios.
13.6 Future Outlook
The phytoremediation technology is still in itsearly development stages and full scale appli-cations are still limited. The study and use ofgenetic modifications must be performed todetermine the true costs and benefits of thistechnology to the ecosystem as a whole. Pro-gress in the field of molecular genetics willallow the analysis of metal hyperaccumulatorplants and should provide new insights intometabolic detoxification processes and identifytolerant genes, thus providing more informationabout the genomes of these model organismsused for this technology.
The genomics can accelerate the discovery ofgenes that confer key traits, allowing theirmodification. In addition, metabolomics canprovide biochemical and physiological knowl-edge about network organization in plants sub-ject to toxic metal stress, providing a much more
238 M. Baunthiyal
detailed understanding of the molecular basis ofhyperaccumulation.
Identification of many signaling pathwaysand proteins within the stress response networkcan contribute to the cellular stress response.The development of DNA and RNA microarraychip technologies in systematic genome map-ping, sequencing, functioning, and experimen-tation may allow the identification andgenotyping of mutations and polymorphisms.This will provide a better insight into structure–function interaction of genome complexityunder toxic metal stress. For example, Mitogen-Activated Protein Kinase (MAPK) pathways areactivated in response to metal stress, whichencourages new strategies for improving planttolerance to heavy metals and phytoremediation.
Molecular genetics approaches such asinsertion mutagenesis can be used to identifygenes involved in hyperaccumulation. Similarly,transposon tagged plants can be screened toidentify mutants impaired in the ability toaccumulate metals. Recently, considerable pro-gress has been made in identifying plant genesencoding metal ion transporters with importantfunctions in cation transport and homeostasis.
Modern molecular techniques, bioinformat-ics, and computational techniques are effectivetools for detailed structure–function genomeanalysis. Approaches allowing recombinationhotspots to be highlighted will further aid plantbreeding efforts. It is clear that both fundamentaland applied research must be carried out inassociation, since the lack of the basic under-standing will make it difficult to exploit many ofthe recent advances in plant molecular biology.
13.7 Conclusion
It is evident that phytoremediation has benefitsto restore balance to a stressed environment, butit is important to proceed with caution before itis applied on a larger scale. The results alreadyobtained have indicated that the plants areeffective and could be used in toxic metal/organic contaminants remediation. Although itappears to be a widely accepted technology
amongst scientists, engineers, and regulators, itis important that public awareness about it isconsidered. Clear and precise informationshould be made available to the general public toenhance its acceptability as a global sustainabletechnology.
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14Future of Biotechnology Companies:A Global Perspective
Sunita Chauhan and Pradeep Bhatnagar
Abstract
In the current scenario of environmental friendliness, need of sustainabledevelopment, globalization, and fast technological developments, bio-technology which has been used in a crude manner by mankind sincethousands of years, has become quite relevant especially in its mostmodern form. Out of the three forms viz. white, red, and greenbiotechnology, white or industrial biotechnology has become thetechnology of the day. The present chapter briefs about the global andregional drivers of its developments all over the world. With an overviewof the global biotechnology clusters, challenges involved in settingbiotechnological companies are discussed. Finally, the great marketpotential involved for biotechnological companies has been highlightedwith a suggestion of three groups of companies (http://www.oecd.org/sti/biotech/44776744.pdf) that can be set up for harnessing the vastpotential involved in biotech products all over the world.
14.1 Introduction
The survival and well-being of any countrydepends on sustainable development. The sus-tainable development is a process of social andeconomic betterment that satisfies the needs andvalues of all interest groups without foreclosing
future options. For this, we must ensure that thedemand on the environment from which wederive our sustenance, does not exceed its car-rying capacity for the present as well as futuregenerations.
The Chemistry Nobel Laureate of 2000, AlanG. MacDiarmid during his lecture on ‘‘TheWorld is Becoming Smaller’’ at the Fifth APECResearch and Development (R&D) Leaders’Forum on March 11, 2004 at New Zealand,brought out the fact that the world population in2003 was 6.3 billion, and by 2050, it would be10 billion. Further the humanity’s top ten chal-lenges for the next 50 years have been listed as:(1) Energy, (2) Water, (3) Food, (4) Environ-ment, (5) Poverty, (6) Terrorism and war,(7) Disease, (8) Education (9) Democracy and
S. ChauhanKumarappa National Handmade Paper Institute(KNHPI), Jaipur, Rajasthan, Indiae-mail: [email protected]
P. Bhatnagar (&)Dean, Life Sciences, The IIS University,Mansarovar, Jaipur, Indiae-mail: [email protected]
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7_14, � Springer India 2014
241
(10) Population. Our best hope of overcomingthese challenges is with new technologies likeBiotechnology (Lim 2009).
14.2 Role of Biotechnologyin Sustainable Development
Biotechnology has been used for more than6,000 years for lots of interesting and practicalpurposes: making food such as bread and cheese,preserving dairy products, and fermenting beer.Although we do not always realize it, biotech-nology is a huge part of our everyday lives, fromthe clothes we wear and how we wash them, thefood we eat and the sources it comes from, themedicine we use to keep us healthy and even thefuel we use to take us where we need to go,biotech already plays, and must continue to play,an invaluable role in meeting our needs. Noother industry is better placed to enhance qualityof life and respond to society’s Grand Chal-lenges of tackling an aging and ever increasingpopulation, healthcare choice and affordability,resource efficiency, food security, climatechange, sustainability, and energy constraints.
The ‘‘new biotechnology’’ that was started inthe early 1970s with direct manipulations of thecell’s genetic machinery through recombinantDNA techniques has fundamentally expandedthe utility of biological systems with its appli-cation on an industrial scale. Scientists andengineers can now confer new characteristics tomicrobes, plants, and animals by changing theirgenetic make-ups. Just like inanimate matters,some biological molecules can now be biologi-cally manufactured or biofactured. The newbiotechnology, combined with the existingindustrial, government, and university infra-structure in biotechnology and the pervasiveinfluence of biological substances in everydaylife, has set the stage for unprecedented growth inproducts, markets, and expectations (Lim 2009).
Biotechnology which is divided into three subfields—red, white (grey) and green biotechnol-ogy— has been very useful for the mankind. RedBiotechnology is used for manufacturing productslike insulin and vaccines for medical uses and in
reproductive technologies like in vitro fertiliza-tion, DNA profiling, forensics, and transplantationtechnologies. White Biotechnology that dealswith creating useful chemicals for the industrialsector through molds or yeast has proven to be ofimmense benefit environmentally in cleaning oilspills and in storing DNA samples of endangeredspecies for future research, besides being usefulfor removing excess nutrients in soil and water andfor detection of landmines. Green Biotechnologydeals with the use of environmental friendlysolutions as an alternative to traditional agricul-ture, horticulture, and animal breeding processes.
Today, Biotechnology is perceived as a rev-olution throughout the world. Scientists, throughR&D have already developed and are continuingto develop cures for diseases that have affectedpeople for decades and even centuries. Simi-larly, certain crops have been developed that canwithstand the brutalities of weather changes,helping poor farmers of the developing countriesto retain their yield, and increase their outputmanifold. Biotechnology can make agriculturemore competitive and sustainable by creatingnew non-food markets for crops. It can helpindustry increase its economic and environ-mental efficiency (eco-efficiency) and sustain-ability while maintaining or improving itscompetitive advantage and ability to generategrowth. Biotechnology, considered a boon bysome, provides great hope to many around theworld, and its benefits will be realized by moreand more people over the years.
14.3 Industrial Biotechnology
Industrial biotechnology has tremendous poten-tial to improve industrial production along allthe three dimensions of sustainable developmentviz. the Society, the Environment, and theEconomy. The application of biotechnology tothe eco-efficient production and processing ofchemicals, materials and bioenergy, and Indus-trial biotechnology as such is a cornerstone ofthe knowledge-based bioeconomy. It utilizes theextraordinary capabilities of microorganismsand enzymes, their diversity, efficiency, and
242 S. Chauhan and P. Bhatnagar
specificity to make products in sectors such aschemicals, food and feed, pulp and paper, tex-tiles, automotives, electronics, and cruciallyenergy. Biological processes are generally moreenvironmentally benign than industrial chemicalprocesses as they take place at low temperatureand pressure, have lower energy input require-ments and lower greenhouse gas (GHG) emis-sions. Also, the raw materials for production arerenewable agricultural feed stocks. Thus,according to Organisation for EconomicCo-operation and Development (OECD), Bio-technology, probably more than any other tech-nology, offers full or partial solutions to majorsocietal problems like healthcare, environmentaldegradation, food security and safety, andenergy supply (OECD 2011).
14.4 Major Drivers of IndustrialBiotechnology
The fast growth of Industrial Biotechnology allover the world is clearly linked to the globalchallenges of climate change, energy security,and the financial crisis. This ‘‘Green Growth’’challenge is faced by developing and developedcountries alike (OECD 2010). There are manyglobal as well as regional drivers of industrialbiotechnology as discussed below:
14.4.1 Global Drivers
14.4.1.1 The Fast TechnologicalDevelopment within IndustrialBiotechnology
Progressing at an enormous rate include devel-opment of basic technologies (e.g., directedevolution, genetic modification tools), develop-ment of tailor made and high performanceenzymes (especially in the number of availableenzymes), improvement of efficiency throughnew developments in reactor and process design,implementation of new applications for existingand new enzymatic systems, and increasingproduction of high-value products, such as
nutraceuticals, cosmaceuticals, and performancechemicals.
14.4.1.2 The Need for SustainableDevelopment
With the growing awareness all over the word,consumers are increasingly conscious about theimpacts of their consumption and choosingproducts with low negative impact on the envi-ronment (e.g., low-carbon economy). Suchenvironmental and social considerations havecreated the need for a switch to clean and sus-tainable processes using renewable resources.Added to the converging economic and envi-ronmental dilemma is the increase in globalpopulation and per capita income. Populationexplosions are resulting into a huge impact onland use and water resources, increased wasteand waste water production, and impact on foodprices through growing demand (OECD 2011).Therefore, need for a sustainable development isone of the major global drivers of industrialbiotechnology.
14.4.1.3 Ongoing GlobalizationGlobalization is acting as a general driver. Thechallenge from the Asian chemical industry iscausing the European Union (EU) and the UnitedStates to look at industrial biotechnology asmeans of sustaining competitiveness. Globalcompetition drives companies to biotech routes,especially in Europe and the USA. Reduction oftrade barriers toward global biotech markets isimportant aspect which cannot be ignored.Another aspect of globalization is that feed stocksand their costs vary across the globe, for example,feed stocks for bio fuel production include sugarcane in Brazil, maize in United States, wheat inEurope, and palm oil in Indonesia.
14.4.2 Regional Drivers
There are regional differences in the drivers forthe development of industrial biotechnology.
14 Future of Biotechnology Companies: A Global Perspective 243
14.4.2.1 The US Top-Down ApproachThe widening ban on the use of the contami-nating and potentially carcinogenic methyl ter-tiary- butyl ether (MTBE) as a gasolineoxygenate necessitates an alternative and etha-nol is gaining share (LoGerfo 2005). Apart fromthe use of ethanol as a fuel, its use as a fueladditive is itself a billion-dollar market. But themuch larger issue and driving force in the UnitedStates is the growing concern over energysecurity and independence which led them tomake vast investments in bioethanol develop-ment. The US industrial biotechnology drive hasbeen led centrally, initiated by Government and/or the administration with massive public fund-ing (Lorenz and Zinke 2005). A further dimen-sion of it is the need of regeneration of the ruralenvironment because according to United statesDepartment of Agriculture a huge number ofagricultural jobs have been lost over the yearsowing to increased efficiency (USDA 2010).
14.4.2.2 The EU Chemical Industryand a Bottom-Up Approach
In the EU the drivers are different. The devel-opment of industrial biotechnology in the EU hasbeen derived from the desire of the chemicalindustry to remain competitive. Data for the10 years from 1999–2009 indicate that the EUhas been the clear leader in terms of worldchemical sales, but it has gradually lost ground toAsia, principally owing to the rise of China. Still,EU exports of chemicals in 2009 accounted for46 % of global chemical exports. (Hadhri 2010).Nonetheless, the threat to its position from Asiais indubitable due to high production costs. Therole of SusChem, the European TechnologyPlatform for Sustainable Chemistry1 is toenhance the European Chemical industry, andindustrial biotechnology is one of its key strate-gic areas. Overall, the EU approach to industrialbiotechnology has been bottom-up, motivated bythe chemical industry. Europe has developed avision for 2025 as they feel that development and
use of industrial biotechnology is essential to thefuture competitiveness of European industry andprovides a sound technological base for the sus-tainable society of the future (Moll and Tanda2012). In general terms, the United States hasfocused on biomass-based energy supply andbulk chemicals, whereas the EU has focusedmore on the manufacture of novel, high-marginproducts. The EU has also been relatively moreconcerned with the environmental impacts, e.g.,compliance with demands for Kyoto protocol.
14.4.2.3 Biomass Utilization in JapanJapan is developing a national strategy for bio-technology, which it sees as a transformativetechnology of strategic importance (Lynskey2006). In March 2006, the Japanese Governmentrenewed the biomass Nippon Strategy of 2002,approved by the cabinet to promote biomass uti-lization and to implement new measures (Kiyoshi2006). The emphasis is on the greater use oftransport biofuels and acceleration of the creationof biomass towns, local municipalities withinfrastructure for utilizing biomass. Owing tolimited agricultural resources and the food versusfuel debate, Japan is to focus strongly on biofuelsderived from cellulosic or other materials whichdo not compete with food supply (USDA ForeignAgricultural Service 2009). They have set a 2030goal of producing biofuels equivalent to 10 % ofdomestic fuel consumption from cellulosicmaterials such as rice straw, wood, and resourcecrops such as sugar cane and sugar beet.
14.4.2.4 The Rise of AsiaThe Asian chemical industry as a whole hasovertaken the EU in terms of sales, and there aregood prospects for the use of biofuels as transportfuel in developing countries. Most of these coun-tries face severe energy insecurity and have largeagricultural sectors able to support production ofbiofuels from energy crops (Liaquat et al. 2010).Population and GDP growth and environmentaland social pressures in developing countriescould be significant drivers for competiveness inindustrial biotechnology. The literature reveals1 (www.suschem.org)
244 S. Chauhan and P. Bhatnagar
the depth and breadth of industrial biotechnologyresearch and innovation in Asian countries such asChina (Zhang et al. 2011); India (Gupta et al.2008); Japan (Anazawa 2010); Malaysia (Hassanand Yaacob 2009); Chinese Taipei (Lin et al.2010); Thailand (Hniman et al. 2011); andVietnam (Thanh et al. 2008).
14.4.2.5 China as a World ForceChina has clearly signaled its intention to be aworld force in industrial biotechnology, with afocus on biofuel. Drivers include a long history ofexpertise in fermentation, a desire for energysecurity and rapidly increasing energy consump-tion, volatility of fossil fuel prices, environmentalconcerns, and providing farmers with an addi-tional income stream to support rural areas(Nesbitt 2009). China is the world’s third largestproducer of ethanol. Existing biobased productionincludes vitamin C and citric acid, and the Chinesechemical industry is making increasing use ofindustrial biotechnology, particularly in biopoly-mers. China is mapping out a 5-year developmentplan (2011–2015) to further concentrate on itsbioindustry and raise its international profile.
14.5 Endeavor of SettingBiotechnology Companiesand Global BiotechnologyClusters
One of the greatest challenges for researchers isconverting scientific discoveries and innovationsinto successful biotechnology companies. Tosucceed, the spin-off phase of biotechnologycompanies has to be crossed by bioentrepreneursand venture capitalists, reluctant to invest in earlystage biotechnology companies. Ukropcova andSturdik (2011) have summarized biotechnologycommercialization in the world and identifiedsignificant biotechnology and life sciences clus-ters on the world map.
There are many issues to be addressed whencommercializing biotechnology research. Theobvious lack of pre-seed capital and inadequatefinancial support from government are not
always to blame (Pavlou 2003). In many cases, alack of commercialization skills in the field ofbiotechnology and innovative financial tools canbe the missing factors to capture the significantvalue from the biotechnology laboratories(Nagle et al. 2003). Although growth anddevelopment of biotechnology spin-offs heavilydepends on financial resources, presence ofencouraging environment is a necessary condi-tion (Booth 2006). Bains (2009) have reportedthe world’s leading biotechnology clusters andcompanies to summarize the results of majorbiotechnology commercialization in the world.
Commercialized biotechnology concentratesin biotechnology clusters surrounded by univer-sitiesandlifescienceresearch institutes (Friedman2006). The idea of a cluster is geographic con-centrations of interconnected sectors, building onstrengths, and removing barriers to development.It requires actions and coordination betweengovernment departments, regional economicdevelopment agencies, universities, companies,and others (Sainsbury 1999). Effective technologytransfer is also necessary with a formal legalinfrastructure for university participation andsufficient funds to file patents. The formation ofnew companies requires a business infrastructurein the community, researchers, technology trans-fer professionals, entrepreneurial company foun-ders, scientists, managers to staff the companies,and knowledgeable investors. It takes a wholecommunity to build a biotechnology cluster. Oncebuilt, the cluster can achieve a sustaining life thatstrengthens itself. The world’s biggest clusters areSan Francisco and Boston area in USA, Cam-bridge, and Oxford area in Great Britain and lastlyMedicon Valley and Bio Valley in the continentalEurope. South Pacific Asian and Australian clus-ters are lately emerged but are fast growing areas(Table 14.1).
14.6 Barriers in Biotechnologyand Challenges Ahead
It is becoming a reality in the world that bio-technology and life sciences are the frontier of aknowledge-based society. Biotechnology is a
14 Future of Biotechnology Companies: A Global Perspective 245
unique industry sector where a high failure ratefor companies is considered the norm. Highpriority for earlier stage companies is to securefunding with more dependence on external fac-tors such as governmental support. The laterstage companies, having access to product-derived funds, are more able to build internalresources and expanding to global markets(Vanderbyl and Kobelak 2008). Some of themajor barriers for Biotechnology companies areas below:
1. High investment and production costs aswell as critical raw material prices
(a) Restrictions of biotechnological productionprocesses from an economic viewpoint.
(b) High investments in R&D and processdevelopment.
(c) Massive new investments to build newproduction facilities.
(d) Higher prices for products from biotech-nology processes normally not achievable.
2. Cyclical raw material prices and limitedavailability
(a) Almost 1:1 correlation of crude oil andbiomass prices during the last years so nogeneral cost advantages of biobased routes.
(b) Rising food and feed demand as a criticaldriver and a significant limitation on theuptake of Industrial biotechnology (espe-cially in debates around biofuels, land usehas been a controversial issue).
Table 14.1 World’s biotechnology and life sciencesclusters with high growth competitiveness index2004–2005 (EMBO Reports 2006)
Life sciencesclustersof North America
Seattle, USA
San Francisco, USA
Los Angeles, USA
San Diego, USA
Saskatoon, Canada
Minneapolis/St. Paul/RochesterUSA
Austin, USA
Toronto, Canada
Montreal, Canada
Boston, USA
New York/New Jersey, USA
Philadelphia, USA
Baltimore/Washington, DC,USA
Research Triangle NC, USA
Central America/SouthAmerica
West Havana, Cuba
Belo Horizonte/Rio de Janeiro,Brazil
Sao Paulo, Brazil
Africa Capetown, South Africa
United Kingdom/Ireland
Glasgow-Edinburgh, Scotland
Manchester-Liverpool, England
London, England
Cambridge-SE England
Dublin, Republic of Ireland
Continental Europe Brussels, Belgium
Medicon Valley, Denmark/Sweden
Stockholm/Uppsala, Sweden
Helsinki, Finland
Paris, France
Biovalley, France/Germany/Switzerland
BioAlps, France/Switzerland
Sophia-Antipolis, France
BioRhine, Germany
BioTech Munich, Germany
BioCon Valley, Germany
Middle East Israel
Oceania Brisbane, Australia
Sydney, Australia
Melbourne, Australia
Dunedin, New Zealand(continued)
Table 14.1 (continued)Asia Beijing, China
Shanghai, China
Shenzhen, China
Hong Kong, China
Tokyo-Kanto, Japan
Kansai, Japan
Hokkaido, Japan
Taipei, Taiwan
Hsinchu, Taiwan
Singapore
Dengkil, Malaysia
New Delhi, India
Hyderabad, India
Bangalore, India
246 S. Chauhan and P. Bhatnagar
3. Complex innovation processes as well ascritical social acceptance and regulations
(a) Industrial biotechnology know how mainlyused in the early stages of the value chain.
(b) Specialized companies normally coveringonly a small share of the value-added alongthe value chain.
(c) Combination of ‘‘technology push’’ and‘‘market pull’’ along the value chain.
4. Critical social acceptance and regulations(a) Social acceptance of industrial biotechnol-
ogy is normally high but some regions stillhave rather a low acceptance of geneticallymodified organisms—in the field of geneticengineering considerably more bureau-cracy and legislation have been seen.
(b) Problem accepting green biotechnology,especially in Europe, has a direct impact onindustrial biotechnology.
Growth depends very much on the develop-ment of green biotechnology. Green and indus-trial biotechnology often combines to anintegrated value chain. The growth and successof biotechnology sector depends on a combina-tion of good education, good science, and goodbusiness (Moses 2003). Biotechnology educationand bioentrepreneurship is a long-term issuerequiring a long-term view; it should not beconstrained by short-term funding. The ability totake risks, prior work experience in private firms,and personal experience in cooperating withindustry lead to a positive attitude towardswitching to private sector employment orentrepreneurship (Fritsch and Krabel 2010).However, despite numerous initiatives to popu-larize and sell science, it seems the attitude andunderstanding of society toward science andscientists remain lower than expected. Scientists’communication in society comes forward as highpriority and great importance (Baron 2010).
Today, the health care ecosystem and its con-stituents face historic challenges. At a time whenkey stakeholders—payers, pharma companies,biotech firms, and their investors—are increas-ingly resource-constrained, we need R&D para-digms that are several shades more efficient andproductive. With aging populations and rapidlygrowing middle classes in emerging markets,
societies need ways to accelerate cures for ail-ments that are expected to impose huge societalcosts, such as neurodegenerative and chronicconditions. For life sciences companies, this willinvolve different ways of thinking about intellec-tual property and recognizing that in some situa-tions, sharing information may create more valuethan protecting it. Regulators will need to adaptframeworks to allow for drug development para-digms that are flexible and learn in real time. Andultimately, patients will need to willingly sharetheir personal health data, with the recognitionthat they might reap some of the biggest dividendsfrom this approach: better health outcomes, betterdrugs and cures for long-intractable diseases.
14.7 Market Potentialfor Biotechnology Companies
Emerging industries such as the life sciences,animal health, agricultural biotechnology, andenvironmental products offer both a potential foreconomic growth and improvements in qualityof life, the environment, and industrial produc-tivity. Even governments in developing coun-tries and investors are seeking to create andenhance biotech entrepreneurship face. Severalenabling trends include increasing numbers ofscience graduates worldwide, accelerating paceof scientific advancement, dominating role ofglobalization enabling greater collaboration, andthe relentless competitive pressure to innovate(Thorsteinsdótti et al. 2004). The largest marketpotential lies in the production of fine chemicalsfor the pharma and agro industry, biopolymersand biofuels (Table 14.2).
The potential involved with Biotechnologycompanies and their future prospects may be fur-ther understood from the following requirements.
14.7.1 Move from Fishingto Aquaculture
A 1997 United Nations estimate indicates thatthe supply of seafood products would have toincrease sevenfold to meet the worldwide
14 Future of Biotechnology Companies: A Global Perspective 247
requirement for fish and other seafood by theyear 2020. Given the rapid decline in world fishstocks, caused mainly by over fishing, it is clearthat demand can only be met by aquaculture.
Worldwide aquaculture production of manyfish species faces several challenges. Amongthem are the selection and supply of suitablebroodstock, growth rate, feed conversion effi-ciency, feeding costs, control of the reproductivecycle, and disease protection. Traditionally, thebroodstock is selected by cross breeding toenhance the fishes’ desired traits. Cross breedingis generally time-consuming and traits are slow toemerge and unpredictable. Gene transfer tech-nology—identifying genes responsible for desir-able traits using molecular biology and thentransferring them to the broodstock—is animprovement over traditional selection andbreeding methods. New traits not present in agenome can also be genetically transferred into anunrelated species, enabling the production of newand beneficial phenotypes. Thus biotechnologycompanies have a great scope in Aquaculture.
14.7.2 From Green Revolutionto Agricultural BiotechRevolution
The Green Revolution of the 1960s depended onagrochemicals and achieved a doubling of pro-duction with only a 10–20 % increase in the
amount of land under cultivation. The worldpopulation is exploding, while one-third of allcrops are still lost to pests and diseases. As theworld population rises to 10 billion over thetwenty-first century, the green revolution willsoon reach its saturation point. Its impact on theenvironment is also increasingly unacceptable.Sir Robert May, Chief Scientific Adviser to theBritish government, warned, ‘‘…we will not beable to feed tomorrow’s population with today’stechnology. We will now need to create cropsthat are shaped to the environment, with bio-technology, whereas before, with the GreenRevolution, our environment was shaped bycrops that were created with the use of chemicalsderived from fossil fuels.’’
Metabolic or nutritional genomics is usinggenetic engineering to improve nutritional valueof plants. Rice, though a staple food for half theworld’s population, is a relatively poor source ofmany essential nutrients, including vitamin Aand iron. An estimated 124 million childrenworldwide are deficient in vitamin A, and aquarter million in Southeast Asia go blind eachyear because of the deficiency. Improved nutri-tion can prevent 1–2 million deaths a year.
The International Rice Research Institute(IRRI) have succeeded in splicing three genes intorice to make it iron-enriched and four other genesto make it rich in b-carotene, a source of vitaminA. The iron-enriched rice and vitamin A-enrichedfunctional rice are combined by crossing. Thus
Table 14.2 Market potential involved and the major trends
S.No. Sector Major trends
1. Fine chemicals • Growing importance of chiral-active pharmaceutical ingredients
• Cost reductions through simplified synthesis paths (especially if molecules are complex)
2. Polymers • Stronger use of renewable raw materials due to cost reasons
• Access to polymers with new properties (e.g., biodegradability)
• Increasing regulatory pressure to realize sustainable solutions (e.g., for packagingapplications)
3. Specialtychemicals
• Strong growth in industrial enzymes for more and more applications
• Increasing advantages of enzymatic processes especially in the food, cosmetic,textile, and leather industries due to customer requirements
4. Base chemicalsand intermediates
• Stronger use of renewable raw materials due to cost reasons
• Cost advantages of biotech processes due to the stricter regulatory environment
5. Biofuels • Increasing advantage due to rising oil prices and climate change
• Significant progress in production technologies with increasing cost competitivenesswithout subsidies
248 S. Chauhan and P. Bhatnagar
biotechnology companies may play a veryimportant role in the Agro-biotech revolution.
14.7.3 Increasing Roleof Biotechnology in Pharmas
There are many other actively researched biotechareas. But a recurring confusion is the boundarybetween biotechnology and pharmaceuticalindustry. To be fair, the boundary is fuzzy. Afterall, of the more than 300 publicly tradedbiotechnology companies, Amgen ($69 billionin capitalization) and Genentech ($55 billion incapitalization), both in California, account forabout a third of the market capitalization.
Genentech, which essentially started thebiotechnology industry in 1976, introduced threedrugs in 2003. Chief among them is Avastin forcancer treatment. Amgen, founded 4 years afterGenentech, has relied on its two runaway mul-tibillion-dollar blockbusters introduced around1990: Epogen, a red blood cell booster to treatanemia; and Neupogen, a white blood cellbooster to treat side effect chemotherapy. Gen-erally speaking, biotechnology companies haveconcentrated on using genetic engineering tomake proteins that can be used as drugs. Phar-maceutical companies, on the other hand, tend torely more on drugs made from chemicals.
Stem cells also regarded as ‘‘magic seeds’’since they possess the ability to replicate indefi-nitely and morph into any kind of tissue found ina human body. They are nature’s blank slates,capable of developing into any of the nearly 230cell types that make up the human body. Poten-tial uses of stem cells are immense. Stem cellscan be used in delineating the complex eventsduring human development. Also, they can beused in the biotechnology and pharmaceuticalindustry to streamline drug safety tests and in celland tissue therapies, and organ transplants.Scientists believe they will lead to cures fordebilitating diseases once thought untreatable.
So, to exploit the vast potential involved inBiotechnology companies, following threegroups of industrial companies can be targetedby policy makers with different approaches:
Dedicated start-ups and small and mediumenterprises (SMEs): provide incentives to fostergrowth based on R&D based innovations.
Diversified SMEs: provide incentives toenable the usage of biotech technologies inestablished production processes.
Dedicated and diversified multinationalenterprises (MNEs): provide incentives to sup-port market introduction and penetration ofbiotech products.
14.8 Conclusion
Keeping in view of the estimated 10 billion pop-ulation of the world by the year 2050 and human-ity’s top 10 challenges, biotechnology can be themost appropriate technology to help us in meetingthose demands and facing all the challenges.
Although the biotechnology industry hasendured several slumps during the last 2–3decades, the overall trends have been positive bymany important measures: number of compa-nies, number of approved products, marketcapitalization and revenues. It is these positivefactors that should help companies with the rightscientific and commercial strategies to prosper inthe future (Kermani and Bonacossa 2003). Thenumber of biotechnology compounds has beenincreasing steadily over the past 20 years,reflecting the key contribution that biotechnol-ogy is now making to healthcare. RecombinantDNA technology has been used to develop anumber of therapeutic proteins, including anti-bodies, cytokines, hormones, and vaccines foruse in tackling and diagnosing a range of dis-orders. Worldwide there are more than 4,000specialized biotechnology companies. The mostwell-known companies are located in the USAand Europe, but there are significant companiesemerging in Canada, Australia, New Zealand,and throughout Asia.
Biotechnology companies are workingeveryday to solve the greatest challenges facingour society whether it is finding a cure forcancer, protecting against bioterror threats, orcreating renewable energy sources, to feed thehungry or to clean our environment. Thus
14 Future of Biotechnology Companies: A Global Perspective 249
biotechnology companies are bound to grow andprosper all over the world. The need is to for-mulate suitable strategies by making national,regional, and internationally harmonized poli-cies, smooth regulatory procedures, and to pro-vide financial and government support so thatthe biotechnology companies may actuallytransform the innovative ideas of today into therealities of tomorrow.
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14 Future of Biotechnology Companies: A Global Perspective 251
About the Editors
Dr. Indu Ravi
Dr. Indu Ravi is presently working as AssistantRegional Director, IGNOU Regional Centre,Jaipur. She has worked as Associate professor inthe Dept. of Bioscience and Biotechnology,Banasthali University, Banasthali (Rajasthan)and has 14 years of teaching experience ofundergraduate and postgraduate classes in thefield of biotechnology. She acquired her postgraduation in Biotechnology from Indian Vet-erinary Research Institute (IVRI), Izatnagar, andher Ph.D. in Biotechnology from BanasthaliUniversity. Her area of specialization is PlantMolecular Biology. She has written one bookand published research papers in National andInternational journals. Email: [email protected].
Dr. Mamta Baunthiyal
Dr. Mamta Baunthiyal has completed herMaster’s degree in Biochemistry fromKurukshetra University, India and her Ph.D. inBiotechnology from Banasthali University,Rajasthan, India. She has 17 years of teachingexperience in undergraduate and postgraduateclasses in Biotechnology. Her research isfocused on the phytoremediation of fluoride andshe has several research articles published inreputed journals. Presently, she is working asHead and Associate Professor in the Departmentof Biotechnology, GB Pant EngineeringCollege, Pauri, Uttarakhand, India. Email:[email protected].
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7, � Springer India 2014
253
Dr. Jyoti Saxena
Dr Jyoti Saxena is a Professor and Head in theBiochemical Engineering Department at BipinTripathi Kumaon Institute of Technology,Dwarahat (Uttarakhand). She has completed herM.Sc. and Ph.D. from Kumaun University,Nainital and her postdoc studies from UNL,USA. She has 19 years of teaching experience inundergraduate and postgraduate classes in Bio-science, Biotechnology, and Biochemical Engi-neering fields. Dr. Saxena has 51 research papersin reputed national and international journals and4 books to her credit. Email: [email protected].
254 About the Editors
Index
AAdipose stem cells, 163AFLP, 28, 29Agarose gel electrophoresis, 27, 74, 95Agricultural biotech revolution, 248Agrobacterium tumefaciens, 2, 216Agro industry, 247Alpha satellite (alphoid) DNA, 9Alzheimer’s disease, 6, 48, 210Amniotic fluid, 157, 159Analyte, 16, 122, 123, 179–181, 183, 184, 186, 189, 190Aneuploidy, 6Antibodies, 48, 51, 53, 79, 84, 87, 117, 121, 122, 135,
138–140, 165, 181, 186, 192, 196–198,200–205, 207–211, 214, 215, 217, 219, 222,223, 249
Antigen, 63, 66, 135, 138, 161, 179, 183, 186, 200,195–204, 208–210, 212–215, 217, 219, 223
Apoptosis, 66, 85, 86, 92, 137Apyrase, 20Arabidopsis, 12, 35, 60, 70, 90, 92, 93, 149, 230, 231,
233Artificial chromosome
bacetrial artificial chromosome (BAC), 1, 5human artificial chromosome (HAC), 1, 8mammalian artificial chromosome, 2P1 derived artificial chromosome, 2P1 phage artificial chromosome (PAC), 1satellite DNA-based artificial chromosome (Satacs), 9yeast artificial chromosome(YAC), 3yeast episomal plasmids (YEPS), 5yeast integrating plasmids (YIPS), 5
Asian chemical industry, 243, 244Autoradiography, 13, 28, 72, 202
BBacteriophage, 2, 6, 7, 11, 141Barriers in biotechnology, 245Bead arrays, 74, 77–79Biochemiluminescent, 20Bioconcentration factor, 228
Bio-entrepreneurs, 245Biolistic
biolistic pressure, 64Biological recognition elements, 179, 180Biomass
biomass utilization, 244Biomedical sequences, 8Biosensor
amperometric, 185applications, 190characteristics, 188electrochemical, 180, 183, 185, 189impedance, 185piezoelectric, 186potentiometric, 185
Biotechnological companies, 241Bloom’s syndrome, 59Brassica, 233Bromodeoxyuridine (BrdU), 108
CCentrifugation, 107, 124, 165Centromere
protein B-binding sequence (CENP-B box), 9Chemokine, 62, 176Chimeric effects, 5Chloroplast engineering, 216, 237Chloroplast transformation, 217, 218, 237Cholera, 207, 210, 212, 223, 225Chromatin, 61, 69, 84Chromatograms, 14Chromatography, 120, 122, 124–128, 130–132, 134,
139–141, 149, 201Clinical trials, 41, 42, 50, 51, 53, 62, 64, 163, 168, 169,
176, 177, 201, 215, 224, 225Cloning
clonal growth , 31, 198efficiency , 1, 2sites, 1, 2vectors, 1
Cognate mRNAs, 58
I. Ravi et al. (eds.), Advances in Biotechnology,DOI: 10.1007/978-81-322-1554-7, � Springer India 2014
255
Conservative biomarkers, 238Contaminants, 74, 129, 186, 227, 235–239Cord blood, 157, 159, 161, 163, 168, 171Cyclodextrin, 64
DDamID, 84Dephosphorylation, 3Diabetes, 68, 117, 136, 147, 148, 164, 167, 171,
172, 223Diversity arrays technology, 36DMSO, 199, 200DNA blots, 28DNA methylation, 57, 78Domains
PAZ domain, 58, 60Piwi domain, 59
Down syndrome, 6, 165Downstream experiments, 2
EEdible vaccine, 207–215, 219, 220, 223–225Electrophoresis
agarose gel electrophoresis, 27, 74, 95capillary electrophoresis, 14, 16, 28denaturing gradient gel electrophoresis, 35, 111electropherogram, 16ethidium bromide, 27, 29polyacrylamide gel electrophoresis (PAGE), 28slab gel electrophoresis, 16
Electroporation, 6, 7, 64, 229Elemental pollutants, 227Endocytosis, 60Endothelial progenitor cells (EPCs), 161Engineered plants, 228Enzymes, 2, 3, 19, 27, 28, 34, 50, 52, 58, 89, 107, 113,
117, 136–138, 147, 180, 181, 183, 184, 186,187, 214, 219, 228, 230, 231, 236, 242, 243
Epigenetic, 55, 58, 84, 85, 137Epistatic effect, 26Epitope, 140, 148, 195, 196, 198, 201–204, 212ESI-MS, 17, 132Exons, 21, 84Extrachromosomal gene delivery, 10Extra-chromosomal retention, 8
FFeeder cells, 199, 200F factor, 5, 6Flanking sequences, 32, 35, 217Flourescence resonance energy transfer
(FRET), 127Foetus, 42Food vaccines, 208Frameshift mutations, 17
GGalistan Expansion Femto Syringe microinjection, 218Gene
alpha-globin gene, 8ampicillin resistance gene, 2antibiotic resistance gene, 3, 112, 236gene augmentation, 42gene chip, 34gene encoding, 50, 169, 223, 230, 231gene ID, 87gene landing efforts, 29gene silencing, 55–58gene tagginggene targeting, 42gene therapy, 8, 10, 41–43, 46, 47, 49–53
Genetically modified organisms, 51, 156, 236Genome
genome complexity reduction, 37genome projects, 7, 12, 18, 112, 133genomic integrity, 55
Global biotechnology clusters, 241, 245Globalization, 243, 247Gluconeogenesis, 68Glycoside bond, 13Green biotechnology, 241, 242, 247Green fluorescent protein (GFP), 60, 69, 108Green vaccine, 208Greenhouse gas, 243
HHaemophilia, 12Halogenated hydrocarbons, 227Haplotype and pedigree analysis, 35Helicases, 59Hematopoietic stem cells (HSCs), 159Hepatitis B
HBsAg , 208, 223Hepatocytes, 52, 66, 168, 170Heteroduplex analysis, 35High-density bead arrays, 78Histone modification, 57, 84Homeostasis, 239Homo neanderthalensis, 12Homologous DNA molecules, 27Human immunodeficiency virus (HIV), 44, 62, 66, 201Hybridization, 27, 28, 34–37, 52, 64, 71–73, 77, 79,
81–83, 87, 88, 94, 95, 107, 183Hybridoma, 195, 196, 198–201, 205Hyperaccumulator, 228, 230, 233, 238Hypercholesterolemia, 51, 63Hypervariable region, 31, 203
IImmobilisation
adsorption, 187covalent binding, 179, 182, 188
256 Index
cross linking, 74, 179, 187, 188entrapment, 186
Immunization, 200, 205, 207, 209, 210, 214, 219, 222Immunoprecipitation, 84, 138, 140In silico, 7, 72, 120, 131, 144In situ polonies, 19In vitro fertilization (IVF), 157Induced pluripotent stem cells (iPSCs), 152, 163Industrial biotechnology, 241–243Inflammation, 47, 48, 88, 176Insertional inactivation, 3Interactomes, 18Introns, 21, 34, 57Isotope coded affinity tag (ICAT), 126
LLabel free tags, 126Laser capture microdissection, 134Lectins, 122, 139, 180, 181, 183Ligase, 3, 19, 79, 80Linkage mapping, 26Lipofection, 9, 49Lung stem cells, 163Lymphocytes, 51–53, 63, 175, 195, 199, 203Lysogenic, 7Lytic, 7, 47
MMalaria, 67, 202, 219MALDI-TOF
MALDI-TOF MS, 16, 17, 122Mammary stem cells, 160Markers
ampicillin resistance gene, 2antibiotic resistance gene, 108codominant markers, 34, 35cos site, 2drug resistance marker , 2PCR based markers, 23, 28
Market potential, 240, 247Mass spectrometry, 16, 17, 35, 117Measles, 69, 207, 219, 220Memory cells, 219Mesenchymal stem cell, 156, 159, 160, 170, 174Metagenomic
application, 110, 112construction, 10enrichment methods, 108library, 10, 19, 27, 32, 107–110, 112, 113PCR, 12, 19, 30screening, 12, 32, 108, 110, 114
Metal-hyperaccumulating plants, 229Metallothioneins, 230, 231Metastasis, 51, 86, 146, 168Micro RNA (miRNA)
agronaute, 55, 61dicer, 55, 57RISC, 55, 59, 61
Microarrayapplication, 17, 85carbohydrate, 72cDNA/oligonucleotie, 72, 87, 92, 93cellular/transfection, 72DNA, 36, 71–73electronic microarrays, 74, 81fabrication, 73, 98fusion genes microarray, 85history, 72oligonucleotide-based printed microarrays, 74protein, 84types, 73, 77, 90
Microbiome, 12Microinjection, 6, 64, 65Microsatellite, 23, 31–33Minichromosomes, 8, 9Minimizing ecological risk, 237Minisatellite, 31, 33Mucosal epithelia, 218Monoclonal antibody
affinity, 58, 135, 196–198assay, 197–200effect of multivalence, 198equilibrium constant, 197, 198immunodiagnostic reagent, 195, 201immunoprophylactic agents, 201infectious diseases, 195, 204, 206large scale production, 201monoclonal antibodies, 195–205rate of association, 197rate of dissociation, 197specificity, 198therapeutic applications, 203–204
Morbidity, 67, 160, 168, 170, 175, 176Mucosal epithelia, 218Mucosal immune response, 208, 209Multi-dimentional protein identification technology
(MudPIT), 125Multilocus minisatellite probe, 31Multipotent, 154, 156, 157, 160–163, 173, 174Mutagenesis, 46, 49, 90, 239Mutation, 3, 12, 17, 31, 32, 34, 41, 42, 55, 59, 69, 85, 86,
177, 183Myeloma cells, 195, 196, 199, 200Myofibers, 48
NNeovascularization, 64Neural stem cells, 161, 162Non-isogenic lines (NILs), 30Nuclear magnetic resonance
(NMR), 132
Index 257
OOlfactory adult stem cells, 162Oligopotent, 154, 156Oncogenes, 6, 85, 86, 176Oral vaccines, 208, 214, 218Origin of replication, 2, 3, 7, 10Osteogenesis, 53Osteoporosis, 51
PP1 phage protein, 6PacBio RS, 18Paleogenomics, 112Parvoviruses, 47PAZ domain, 58, 60PEG, 49, 63, 199, 200, 218PEGylation, 49Peptide mass fingerprinting, 122, 131Pesticides, 227Petroleum hydrocarbons, 227Petunias, 56Peyer’s patches, 219Phage coat proteins, 2Pharma industry, 247Pharmacokinetic profiles, 63Phosphodiester bond, 13Phosphorothiolation, 63Phred programme, 15Phylogenetic tree, 34, 112Physical mapping, 3, 29Physical restrictions, 235Phytochelatins, 230Phytoextraction, 228, 231Phytoremediation, 227–231, 234–235Plant metal transporters, 230Pleiotropic effect, 26Pleuripotent, 152, 154, 155Point mutations, 28, 34, 67Polychlorinated biphenyls, 227Polycyclic aromatic hydrocarbons, 227Polymerase chain reaction
allele specific PCR primers, 35amplicon, 32amplicons, 79bridge amplification, 19emulsion PCR, 19, 20IPTG, 7ISSR-PCR technique, 33PCR based markers, 23RAPD, 23real-time (equipment) PCR, 35
Polymerasepolymerase slippage, 31
Positive clones, 32, 108, 109, 198Post transcriptional, 57Post translational modifications, 136Primer, 14, 17, 19, 20, 28–30, 32, 33, 35, 79, 80, 93, 94Promoter sequence, 6
Protein chips, 134, 135Protein Loquacious, 60Proteoinformatics/Bioinformatics, 141Proteomic
applications, 142basic steps, 117, 121classification, 120proteomic complex datecton using
sedimentation, 134techniques, 117, 120, 122, 132, 134
QQuantitative trait locus (QTL), 28
RRabies, 202, 210–212, 214, 222Radioactive label, 13Receptors, 47, 51, 52, 62, 67, 137, 153, 160, 181, 183,
203, 204Recessive alleles, 3Recombinant DNA, 1, 2, 12, 42, 48, 140, 183, 217, 242,
249Red biotechnology, 242Regulatory issues, 207, 224Regulatory sequences, 6Replicons, 1Restriction endonuclease
restriction enzymes, 27–29restriction site-associated DNA, 37restriction sites, 2, 27retrovirus, 2, 43maps, 21
Reverse complement, 57Reverse transcriptase, 45, 66, 161Ribozyme, 43, 50
SScorable phenotype, 1Seed purity testing, 35Selectable marker, 1, 2, 10Sequence characterized amplified regions, 30Sequence-tagged microsatellite site, 32Sequencing
autonomous replicating sequence (ARS), 3, 4contig, 18, 19, 36DNA nanoball sequencing, 18encoding sequence, 6ion semiconductor sequencing, 18massively parallel signature sequencing, 18non-Sanger sequencing rechnologies, 18polony sequencing, 18pyrosequencing, 12, 18–20Roche 454 genome sequencing, 18, 19Scaffold, 18, 167, 174shotgun sequencing, 17, 35, 110, 111solid sequencing, 18, 20, 22
258 Index
whole genome shotgun sequencing, 17, 18, 21, 36,37, 78
Shot gun proteomics, 132Signal transduction, 57, 137Simple sequence repeats, 31, 35Single feature polymorphism, 37Single nucleotide polymorphism (SNP), 34, 85Single primer amplification reaction, 33Single strand conformational polymorphism assays, 35Size fractionation, 3Solid-phase arrays, 73Somatic cells, 41, 42, 53, 159Southern blotting, 27, 28, 72, 73Southern hybridization, 32, 34Spumaviruses, 4416S rRNA, 88, 102, 109, 111Stable genomic integration, 215Stable isotope probing (SIP), 108Stem cell research
applications, 11classification, 97identification, 65, 73, 87isolation, 5, 8, 121, 165resources, 152, 156, 157, 159, 162, 165, 168technology, 157, 158, 166therapy, 151, 159, 161, 166, 167, 171–173, 176
Stress factors, 235Subunit vaccines, 207, 208, 212, 213, 215Suspension bead arrays, 74, 78, 79, 81Symbiosis, 111Synteny, 38Systemic immunity, 213
TTaqman allelic discrimination, 35Telomeres
telomere-mediated chromosome fragmentation, 8Terminase, 6Testicular cells, 162Tiling array, 84, 85TNT, 229, 236Totipotent, 151, 153Transcriptional, 50, 55–59, 68, 88, 90Transcriptomes, 18Transcripts, 12, 45, 67, 71, 117, 119, 136, 171Transducer
acoustical, 185electronic, 179, 180mechanical, 179optical, 179, 180, 186thermal, 186
Transformants, 4Transformation, 6, 7, 97, 141, 173, 215–217, 237Transgenic, 6, 48, 60, 70, 92, 161, 168–170, 207–212,
215, 217, 218, 220, 223–225Transient expression, 216Transposons, 56, 59
Trypanosomes, 56Two-dimensional gel electrophoresis, 120, 122, 131, 147
UUmbilical cord blood stem cells, 163, 168Unipotent, 157
VVariable number of tandem repeats (VNTRs), 31Vaccine
advantages, 212, 213, 216–218, 222applications, 209, 217, 219, 222challenges, 214, 225hepatitis-B, 207–215, 222host plant, 209, 216limitations, 208, 214, 215, 223mucosal immunity, 208, 209, 212, 219production, 208, 209, 213, 215side effects, 210, 213, 215, 225
Vectorsadeno-associated virus, 43, 47, 52adenovirus, 43, 46, 52agrobacterium vector, 229baculo virus, 2capsid, 45, 47, 199, 268copy number, 2cos Site, 2cosmid, 2, 3episomal viral vectors, 10episome, 10fosmid, 1068immunocompetent, 50insert Size , 2, 6lentiviruses, 44, 62liposomes, 49, 63loxp Site, 6oncoretroviruses, 44phage k, 2retroviral vectors, 51vector functions, 1vector Size, 2vector-to-vector ligation, 3virion, 47
Virulence factor, 88
WWerner’s syndrome, 59White (grey) biotechnology, 242White biotechnology, 241
XX-ray crystallography, 120, 122, 133, 141, 149X-ray tomography, 122, 133
Index 259
