Chapter 17

Gene Technology

Protein

RNA

DNA

transcription

translation

CCTGAGCCAACTATTGATGAA

PEPTIDE

CCUGAGCCAACUAUUGAUGAA

Central Dogma: DNA -> RNA -> Protein

Genetic Recombination in Humans

There are three ways in which meiosis and fertilization ensure that a child has a different combination of genes from that of either parent:

1. Independent assortment of chromosomes during metaphase I

2. Crossing-over during prophase I3. Upon fertilization, recombination of chromosomes

from different individuals (via their gametes) occurs.

Recombinant DNA technology or genetic engineering was developed in 1971-1973

Their core was gene cloning Lead to DNA sequencing techniques that

enabled the structures of individual genes to be

determined Lead to procedures for studying the regulation

of individual genes

Genetic recombination - transfer of DNA from one organism

(donor) to another recipient. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms (homologous, non-homologous).

Types Of Recombination

Generalized or Homologous Recombination - Occurs during prophase of meiosis I and involves exchange between homologous strands of DNA

Site Specific Recombination - Short homologous sections of bacterial and phage DNA serve as a site for recombination and thus incorporation of phage DNA into bacterial chromosomes

Transposition - Not truly recombination between different genomes, but the movement of transposons within a genome

Homologous recombination-

homologous DNA sequences having nearly the same

nucleotide sequences are exchanged by means of Rec

A proteins. This involves breakage and reunion of

paired DNA segments as seen in Natural mechanisms

of genetic recombination in bacteria include:

a. transformationb. transductionc. conjungation

The Current Prokaryotic

Recombination Model

内切酶

(recBCD)DNA 侵扰(recA)

分支迁移 (recA)

内切酶(recBCD)

DNA 连接酶

5´ 3´

5´3´5´

3´

5´3´

5´ 3´

5´ 3´

5´3´

5´3´

5´ 3´

5´ 3´

5´3´5´3´

5´3´

5´ 3´

3´ 3´

5´3´

5´3´

3´5´

5´ 3´

5´3´

5´3´

Holiday 中间体

5´ 3´

5´ 3´

5´3´

5´3´

目 录

Holiday 中间体

5´ 3´

5´ 3´

5´3´5´3´

5´3´

5´

5´5´

3´

3´

3´

5´ 5´

5´5´

3´

3´

3´

3´

5´ 5´

5´5´

3´

3´

3´

3´

5´5´

5´5´

3´

3´

3´

3´

5´ 5´

5´5´

3´

3´

3´

3´

内切酶(ruvC)内切酶

(ruvC)

DNA连接酶

DNA连接酶

片段重组

体拼接重组

体

Patch recombinationsplice recombination

Genetic Transfer & Recombination In Bacteria

Three kinds of genetic exchanges between prokaryotes

Three kinds Conjugation

Mediated by plasmids Transformation

Mediated by free DNA Transduction

Mediated by phages

Bacterial Conjugation

Bacterial Conjugation is genetic recombination in which there is a transfer of DNA from a living donor bacterium to a recipient bacterium. Often involves a sex pilus.

The 3 conjugative processes

I. F+ conjugation

II. Hfr conjugation

III. Resistance plasmid conjugation

F+ Conjugation- Genetic recombination in which there

is a transfer of an F+ plasmid (coding only for a sex

pilus) but not chromosomal DNA from a male donor

bacterium to a female recipient bacterium. Involves

a sex (conjugation) pilus. Other plasmids present in

the cytoplasm of the bacterium, such as those coding

for antibiotic resistance, may also be transferred

during this process.

I. F+ Conjugation Process

Conjugal transfer of plasmid

The 4 stepped F+ Conjugation

1. The F+ male has an F+ plasmid coding for a sex pilus and can serve as a genetic donor

2. The sex pilus adheres to an F- female (recipient). One strand of the F+ plasmid breaks

The 4 stepped F+ Conjugation (cont’d)

3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium

4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/conjugation/f.htm l

F

Transformation

Genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA.

Involves 4 steps

1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA

4. Exchange is complete

The 4 steps in Transformation

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html

Transduction

Genetic recombination in which a DNA fragment is transferred from one bacterium to another by a bacteriophage

Structure of T4 bacteriophage Contraction of the tail sheath of T4

What are Bacteriophages?

Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria

An infection

cycle

Transduction

There are two types of transduction: generalized transduction: A DNA fragment is

transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.

specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle

Site Specific Recombination

Short homologous sections of bacterial and

phage DNA serve as a site for recombination

and thus incorporation of phage DNA into

bacterial chromosomes

Integration of Lambda DNA-overview.

att = attachment site

INT = integrase

O = center core of 15 bases = the same in phage & bacterial

B,P = different in size and sequence in bacterial & phagedsDNA

XIS = Excisionase

The control of INT & XIS activity determines it latency or not.

Integration of Lambda DNA-Detail of crossover

例 : 细菌的特异位点重组

沙门氏菌 H 片段倒位决定鞭毛相转变

Transposition

-Movement of gene to a new site, on same or a different chromosome

Does not require extensive homology

Transposable elements

Insertion sequence

插入序列的复制性转

座

目 录

Transposons: Mobile genetic elements that enable genes to move between non-homologous sites in DNA –

Transposable elements. ⋅Altered expression of genes in new environments

Gene technology

A set of methods and techniques used to study biological processes on the molecular level

There have been considerable developments in this field during the past two decades

Eg: new and powerful ways for the isolation, analysis, and manipulation of nucleic acids

What is gene cloning

(1) A fragment of DNA is inserted into a circular DNA molecule

called a vector, to produce a chimera or recombinant DNA

molecule

The basic steps in gene cloning experiment are as follows:

(2) The vector acts as a vehicle that transports the gene into a

host cell

(3) Within the host cell the vector multiplies, producing

numerous identical copies

(4) When the host cell divides, copies of the recombinant DNA molecule are passed to progeny and further vector replication takes place

(5) After a large number of cell divisions, a colony, or clone, of identical host cells is produced

Each cell in the clone contains one or more copies of the recombinant DNA molecule

The gene carried by the recombinant molecule is now said to be cloned

The basic steps in gene cloning

Why gene cloning is so important

Gene isolation by cloning Cloning allows individual fragments of DNA to be

purified isolated long genes or those that have never been studied

before

This technique can provide a pure sample of individual gene, separated from all the other genes in the cell

Cloning allows individual fragments of DNA to be purified

Clone A large population of identical molecules, or cells that arise from a common ancestor.

Cloning

The basic concepts about gene cloning

The process that produces a large number of DNA or cell copies

vector target DNA Recombination DNA

Transformation

bacteria Recombination DNA

amplification

Transfer the bacteria to

a solid culture plate

Screening the bacteria containing

the recombinant DNA

The process of gene cloning with the

plasmid as vector

DNA recombinationThe process that two DNA molecules from different source join together by covalent bond to form a new DNA molecule is called DNA recombination. Recombinant DNA

DNA recombination technique By the application of some tool enzymes, the target gene and vector are ligated together, then introduced into the recipient cells which multiply and express the protein products coded by the target gene, that is, DNA recombination technique, DNA cloning or Gene cloning, Molecular cloning.

Genetic engineering

All the work or methods used related to

the gene cloning and the target gene expressed

in host cells to produce the special protein or

polypeptide, or to change the character of an

organism, are called genetic engineering.

The requirements for the gene cloning

(1) The target genes

(2) The vectors

(3) The tool enzymes

(4) The host cells

Target DNA

• cDNA

• Genomic DNA

The cDNA are synthesized by reverse transcriptionase based on their mRNA templates of a cell line or tissue.

It represents whole DNA sequence of a genome

Purification of DNA from living cells

Preparation of total cell DNA (RNA)

Preparation of plasmid DNA

Preparation of bacteriophage DNA

Cloning vector——Cloning vectors are DNAs which can carry target genes, transfer them into the recipient cells.

Cloning vector classes

Plasmid DNAPhage DNAVirus DNA

Vectors

As for the expression vectors, they can make the proteins which are coded by the target gene expressed in the host cell

Plasmids Basic features of plasmids

Small (less than 10kb), Circular, duplex molecules of DNA

Exist at low or high copies within the bacteria, but useful plasmid present in multiple copies

Replicate independently from the bacterial cell

Contain selectable markers, eg: the antibiotic resistance capability conferred to bacterium

Possess at least one DNA sequence that act as an origin of replication Multiple RE sites ( multiple cloning sites, MCS)

plasmid ( 质粒 )

Fig pBR322 Go to pBR322

b

Fig pUC19 Go to pUC

Origin of replication

Multiple cloning site (MCS)

Lac Z β-galactosidase gene

Ampicillin resistance gene

Bacteriophages

Bacteriophages, or phages are viruses that specifically infect bacteria

Simple in structure, merely of a DNA (or occasionally RNA) carrying genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein

Basic features of bacteriophages

The general pattern of infection

Attaches to the outside of the bacterium and injects its DNA chromosome into the cell

The phage DNA is replicated, usually by specific phage enzymes coded by genes on the phage chromosome

Other phage genes direct synthesis of the protein components of the capsid, new phage particles are assembled and released

Lytic cycle:

With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 min. This type of rapid infection is called lytic cycle.

Lysogenic infection:

Characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions

The result of Infection

Common used phages

Bacteriophage λ

A linear dsDNA approximately 49 Kb in lengthAfter infection it forms circular structures The phage DNA is inserted into the bacterial genomeThe first two classes of vector to be produced were λ i

nsertion (λgt phages) and λ replacement (EMBL phage

s)

Bacteriophage M13

A circular ssDNA, and has been used for sequencing of a cloned target DNA fragment

Fig pUC19 Go to pUC

Origin of replication

Multiple cloning site (MCS)

Lac Z β-galactosidase gene

Ampicillin resistance gene

Other vectors

Cosmid ( 粘性质粒 )

Bacterial artificial chromosome (BAC) and yeast chromosome

Viruse are used as vectors, eg: retro-virus, adeno-virus, adenoassociated virus, etc

Other vectors

The tool enzymes

Nucleases—cut, shorten or degrade nucleic acid molecules

Ligases—join nucleic acid molecules together Polymerase—make copies of molecules Modifying enzymes —remove or add chemical gro

ups Topoisomerases —introduce or remove supercoils

from covalently closed-circular DNA

Nucleases degrade DNA molecules by breaking the phosphodiester bonds

There are two different kinds of nucleases

Exonucleases remove nucleotides one at a time fr

om the end of a DNA molecule

Endonucleases are able to break internal phospho

diester bonds within a DNA molecule

ligases

To repair single-stranded breaks(discontinuities) that arise in double-stranded DNA molecules during DNA replication

Join together two individual fragments of double-stranded DNA

Polymerases Synthesize a new strand of DNA complementary

to an existing DNA or RNA template Four types of DNA polymerase are used routinely

in genetic engineering

DNA polymerase I: from E.coli. Synthesizes dsDNA by formation of

a 5’,3’-phosphodiester bond

Klenow fragment : removes the first 323 amino acids from DNA poly

merase I , Synthesizes DNA by formation of a 5’,3’-phosphodiester

bond

Reverse transcriptase: synthesizes DNA from RNA template

Taq DNA polymerase: used in the PCR, it is the DNA polymerase I f

rom bacterium Thermus aquaticus

DNA modifying enzymes

Alkaline phosphatase from E.coli, calf intestinal tissue or arctic shrimp removes the phosphate group present at the 5’term

inus of a DNA molecule

Polynucleotide kinase from E.coli infected with T4 phageHas the reverse effect of alkaline phosphatase, adding phosphate groups onto free 5’

termini

Terminal deoxynucleotidyl transferase from calf thymus tissue adds one or more deoxyribonucleotides onto the 3’ t

erminus of a DNA

DNA modifying enzymes

Topoisomerases

Change the conformation of covalently closed-circular DNA by introducing or removing supercoils.

Enzymes for cutting DNA-restriction endonucleases

The initial observation that led to the eventual discovery of restriction endonucleases (RE) was made in the early 1950s

Restriction occurs because the bacterium produces an enzyme (called restriction endonucleases) that degrades the phage DNA

The discovery of these enzymes led to Nobel prizes for W.Arber, H. Smith and D. Nathans in 1978

Three different classes of RE are recognized, but the most important one is RE II which is used in DNA manipulation

Type II restriction endonucleases (RE) cut DNA at specific nucleotide sequences

Generally, 4~8 bases be found, mostly 6 bases, a few of 8~10 bases

The sequences discriminated usually are palindrome structure

To cut the double strands of DNA at special sites and to yield two kinds of ends: blunt ends and sticky ends

Blunt ends and sticky ends:

Sticky or cohesive ends:

the cleavage is staggered by two or four nucleotides

the resulting DNA fragments have short single-strand

ed overhangs at each end

Base pairing between them can stick the DNA molec

ule back together again

Restriction endonucleases with different recognition s

equences may produce the same sticky ends eg: Bam

H I (GGATCC) and Bgl II (AGATCT)

5’-GGTGAATTCAGC…-3’3’-CCACTTAAGTCG…5’

5’-TTGCTGCAGAAG…-3’3’-AACGACGTCTTC…5’

5’-sticky end (EcoR I )

3’-sticky end ( Pst I )

5’-GGTG AATTCAGC…-3’3’-CCACTTAA GTCG…5’+

5’-TTGCTGCA GAAG…-3’3’-AACG ACGTCTTC…5’+

blunt end or flush end

5’-CCCGGG…-3’3’-GGGCCC…5’

5’-CCC GGG…-3’3’-GGG CCC…5’+

*The ligation efficiency between the blunt ends is not as high as that of the stickly ends.

Sma I

Make a simple double-stranded cut in the middle of the recognition sequence

Naming of RE

Escherichia coli RY13 I

EcoR I

The genus name of bacteria

The species name of bacteria

The strain name of bacteria

The order of the RE found in bacteria

REs are usually named after the bacterium from which they are isolated.

The requirements for the gene cloning

(1) The target genes

(2) The vectors

(3) The tool enzymes

(4) The host cells

The basic process of recombination technique

* The preparation of target DNA* The selection and preparation of vectors

* The ligation of DNA fragments in vitro

* Foreign DNA be transported into host cells

* The screening and identifying of target DNA

1. The preparation of target DNA

(1)To prepare from genomic library genomic library contains a comprehensive DNA

fragments from genomic DNA cut by the specific RE.

During the construction of the genomic library, the DNA fragments and their vectors are ligated, and then introduced into the recipient cells.

It represents whole DNA sequence of a genome

(2) To prepare from cDNA library or cDNA

Extracting total mRNA Reverse transcription

Ligation introduction

It represents the population of mRNAs coding for gene and protein expression

(3) To prepare the gene fragment with

other methods

1) PCR amplification

2) To synthesize the DNA fragment by

chemical method

it is typically used for those of

the small biologically active peptides

2. The selection and preparation of vectors

Plasmid λ phage cosmid M13 phage

Capacity < 10 kb < 22 kb 40~50 kb < 1 kbof cloning

gDNA library - + + -

cDNA library + + - -

Subcloning + - - +

Sequencing + + - +

E coli expression + + - -

3. Construction of Recombinant Molecules

Both purified DNA fragments and vectors are digested with the same restriction enzyme to give complementary cohesive ends

•Analyzing the result of restriction endonuclease cleavage

Separation of molecules by gel electrophoresis Visualizing DNA molecules in a gel (EB staining) Comparison with size markers

joining together of the vector molecules and DNA

to be cloned

The enzyme that catalyses the reaction is called D

NA ligase, which purified from E.coli bacteria that

have been infected with T4 phage

•Ligated by T4 ligase to recombinant molecules

CTTAAGGAATTC

EcoR Ⅰ

CTTAAGGAATTC GAATTC5’CTTAAG 5’3’

3’ EcoR Ⅰ

CTTAAAATTC G5’

G 5’3’3’

GCTTAA

AATTCG

5’3’

5’ 3’

CTTAAG CTTAAGGAATTC GAATTC

ligationCTTAAG CTTAAGGAATTC GAATTC

Bidirection insertions

(1) Sticky-ended ligation

VectorTarget gene

Restriction endonucleases

Restriction endonucleases

T4 DNAligase15ºC

recombinateSelf-ligated vector

Self-ligated target gene

(2) Blunt-ended ligation

4. Introduction of DNA into living cells Serves two main purposes:

allows a large number of recombinant DNA molecule

s to be produced from a limited amount of starting ma

terial

Purification

-Methods Transformation

Transfection

Infection

Transformation ----The uptake of DNA by bacterial cells preparation of competent E.coli cells

50 mM CaCl2 is tranditionally used.

Another alternative is by electroporation

• In recent years, transformation has been extended to include uptake of any DNA molecules by any type of cell

Whether the uptake results in a detectable change in the cell

Whether the Cells involved is bacterial, fungal, animal or plant

Introduction of phage DNA into bacterial cells

Two methods:

Transfection

purified phage DNA, or recombinant phage molecules, is mixed with competent E.coli cells and DNA uptake induced by heat shock

Transfection

Introduction of phage DNA into bacterial cells Two methods:

In vitro packaging single strain system: the defective λphage c

arries a mutation in the cos sites two strain system: two defective λphage carr

ies a mutation in a gene for one of the components of the phage protein coat

Phage infection is visualized as plaques on the agar medium

In vitro packaging

The problem of selectionA restriction digest of total cell DNA produces not

only the fragment carrying the desired gene, but also many other fragments carrying all the other genes

Numerous different recombinant DNA molecules are produced

A variety of recombinant clones are obtained

5. Screening and Identification of Recombinants

Target Genes Carried by Plasmid

1 plasmid1 cellRecombinant

PlasmidTransformation

Target GeneRecombination

Restriction

Enzyme

Restriction

Enzyme

Ch

rom

oso

mal

DN

ATarget Genes

DNA Recombination

TransformationHost Cells

Juang RH (2004) BCbasics

Amplification and Screening of Target Gene

1

1 cell line, 1 colonyX100

X1,000

PlasmidDuplicationBacteria

Duplication

Plating

Pick the colonycontaining target gene

=100,000Juang RH (2004) BCbasics

There are two basic strategies for obtaining the clone you want

Direct selection for the desired gene

the only clones that are obtained are clones of the required gene

There are two basic strategies for obtaining the clone you want

Direct selection for the desired gene

the only clones that are obtained are clones of the required gene

Identification of the clone from a gene library

entails an initial shotgun cloning experiment, to produce a clone library representing all or most of the genes present in the cell, followed by analysis of the individual clones to identify the correct one

Correct clone

A clone library

Direct selection an antibiotic resistance gene

Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation plasmids contain sequence (lacZ) coding

for N-terminal amino acids (α fragment) of β–galactosidase

Mutant cells contain sequence (lacZ) coding for C-terminal amino acids (ω fragment) of β– galactosidase

By αcomplementationThe enzymatic activity is dependent on the

coexpression of the complete fragments, which can hydrolyzes the specific substrate X-gal (5-bromo-4-chloro-3-inolyl-β-D-galactoside) to turn to a blue colored one under the induction of IPTG （ isopropyl thiogalactoside)

The recombinant molecules have no this enzyme activity because the insertion of target gene into the lacZ region disturbs the expression of αfragment, and therefor, the colour of the recombinant molecule containing the colony is white.

Based on this blue-white colony screening

White clone contains the recombinant, but blue clone not contain recombinant

Multiple cloning sites

The sequence coding the N end fragment of β-galactosidase

Ampr

promoter

transformation

Chromosome

The sequence coding the C end fragment of βgalactosidase

The growth of bacteria on the culture with X-gal

The blue clone containing the pUC18

The blue clone containing the pUC18

transformation

Cleavage N end

External DNA

Cleavage N end

Recombinant pUC18

The growth of bacteria on the culture with X-gal

The white clone containing the recombinant pUC18

α-mutual complement screening

Ampr

Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation

Colony/plaque in situ hybridization is used for positive colony screening

Colony Is Screened by Hybridization with Probe

Cover withfilter

paper

Autoradiography

Add probe

Transferring …

Collect filter paper

Dissolve cell DNA denatured

Jua

ng

RH

(2

00

4)

BC

ba

sics

Colony hybridization

Direct selection

an antibiotic resistance gene

Marker rescue ---by αcomplementation

Colony/plaque in situ hybridization is used for positive colony screening

Immunological Technology

Methods for clone identification

Colony PCREnzyme digestionnucleic acid hybridizationDNA sequencing

Using PCR to detect gene targeting events

M 1 2 3 4 5 6 7 8 9 10

1kb

Figure2b.The positive plasmids after cutted by EcoR IM----1Kb DNA ladder1∽10, positive plasmids cutted by EcoR

M 1 2 3 4 5 6 7 8 9 10

Figure2a. The clones of positive plasmidM----1Kb DNA ladder1∽10, positive plasmid

A. before cut with RE

To identify the target gene band after cutted by RE

B. after cut with RE

Identification with restriction enzymes

The basic process of recombination technique

* The preparation of target DNA* The selection and preparation of vectors

* The ligation of DNA fragments in vitro

* Foreign DNA be transported into host cells

* The screening and identifying of target DNA

6. Expression of the cloned gene

Different vectors are selected for cloning

cloning vectors are used for replicating and amplifying genes

Expression vector are applied to express the gene product

Cloning vector

Antibiotics resistance, MCS and screening

Expression vector

Antibiotics resistance, MCS and screening

Contains regulatory sequences for transcription and translation, eg; promoter, SD sequence for 16s rRNA binding, and a terminator which is ρfactor independent

Expression system

Prokaryote expression system E.coli (most popular) Its easy culture, fast proliferation, low

expense, large scale production Lack of the processing capability after

transcription and translation

Notes during expression

Infusion proteins maybe formed when other sequences coding amino acids linked to a target gene are co-expressed together with target proteins

Purified by affinity chromatography, followed by the unnsecesary peptides cut out

eukaryote expression system Mammalian cells are stable and

repeatable can process hnRNA to become mature

mRNA, as well as the post-translation

modifications

Application of Recombination DNA Technology

DNA Recombination Medical Production

产 品 功 能组织胞浆素原激活剂 抗凝血液因子 VIII 促进凝血颗粒细胞 - 巨噬细胞集落剌激因子 剌激白细胞生成促红细胞生成素 剌激白细胞生成生长因子 (bFGF, EGF) 刺激细胞生长与分化生长素 治疗侏儒症胰岛素 治疗糖尿病干扰素 ( 1b, 2a, 2b, ) 抗病毒感染及某些肿瘤白细胞介素 激活、剌激各类白细胞超氧化物歧化酶 抗组织损伤

单克隆抗体 利用其结合特异性进行诊断试验、肿瘤导向治疗

乙肝疫苗 (CHO, 酵母 ) 预防乙肝口服重组 B 亚单位菌体霍乱菌苗 预防霍乱

目 录

Gene diagnosis It is recognized that the abnormal

structure and expression of the gene are involved in the pathogenesis of diseases

Gene diagnosis is the detection of the abnormalities of the candidate genes by ways of molecular biology and molecular genetics

Sickle cell anemia belongs to gene point mutation The 6th codon GAG was changed to GTG Glu is changed to Val Abolish an MstII restriction site which spans codons 5-7

For examples ：

Sequencing HbS proteins revealed a single change: Glu6Val in the β chain. Fiber formation (R) at low [O2] causes sickling of RBCs (center).

×

Mst restriction site (GCTNAGG)Ⅱ

5´ 3´

Normal gene

5´ 3´

Mutation gene

1.15kb

1.35kb

Restriction mapping analysis of sickle cell anemia

++

﹣﹣

0.2kb

1.15kb

1.35kb

Normal Carrier Sickle cell homozygote

镰状红细胞贫血患者基因组的限制性酶切分析

Gene Therapy Gene therapy is the way to transfer

genetic material which exerts the biological function into the cells of patients to treat the disease

Genetic material: normal gene, recombinant DNA, RNA, synthetic oligonucletides

They may integrate into the chromosome or express separately

The Strategies and technologies of gene therapy

Gene correction The abnormal bases of a gene are correct

ed

Gene replacement The defected gene is replaced by the nor

mal one which can integrate into the chromosomes by homologous recombination or remain extrachromosomal

Gene augmentation The target gene is introduced to the defecte

d cells or other cells

Gene inactivation the expression of the gene is intervened to b

lock or inhibit the inappropriate genes in vivo on both transcriptional and translational level

The Strategies and technologies of gene therapy

Applications of Gene Therapy

The first apparently successful application was initiated on Sep. 14, 1990 for ADA deficiency

Results in a lymphopenic form of SCIResults in a lymphopenic form of SCID that is fatal in early childhood.D that is fatal in early childhood.

ADA SCIDADA SCID ( (Severe Combined ImmunodeficiencySevere Combined Immunodeficiency DiseasesDiseases )

Autosomal recessive disorder ADA = adenosine deaminase (an enzyme

of purine metabolism) ADA is an important enzyme in the purinADA is an important enzyme in the purin

e catabolic pathway, catalyzing the irrevee catabolic pathway, catalyzing the irreversible deamination of adenosine to inosinrsible deamination of adenosine to inosine.e.

SCID with ADA DeficiencySCID with ADA Deficiency

ADENOSINE

INOSINE

HYPOXANTHINE

XANTHINE

URIC ACID

GUANOSINE

GUANINE

Purine Catabolism Pathway

AdenosineDeaminase

PurineNucleoside

Phosphorylase

PurineNucleoside

Phosphorylase

XanthineOxidase

XanthineOxidase

Guanase

SCID with ADA DeficiencySCID with ADA Deficiency

The enzyme deficiency inhibits the normal The enzyme deficiency inhibits the normal

catabolism of purines.catabolism of purines.

Results in the accumulation of metabolic suResults in the accumulation of metabolic su

bstrates that are toxic to lymphocytes, particbstrates that are toxic to lymphocytes, partic

ularly in the inhibition of lymphocyte functiularly in the inhibition of lymphocyte functi

on. on.

GENE THERAPY IN ADA SCIDGENE THERAPY IN ADA SCID

ADA deficiency was the first disorder to be treated by gene therapy (Bordignon et al 1995)

The initial targets for genetic manipulation were bone marrow (BM) stem cellsbone marrow (BM) stem cells and peripheral blood lymphocytes (PBLs)peripheral blood lymphocytes (PBLs)

Vectors expressing human ADAcDNA (1.5 kbp) with their own promoters were transfected into BM stem cellsBM stem cells and PBLsPBLs in vitro

6 months after gene therapy ended, vector-derived DNA was found in the PBLsPBLs

GENE THERAPY IN ADA SCIDGENE THERAPY IN ADA SCID

The Cloning Procedure Used for Creating Dolly