Cloning Technique

7/25/2019 Cloning Technique

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CLONING TECHNIQUE


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What is cloning ?

Cloning is a process that

create an exact genetic

replica of another cell,tissue or organism.

Copie material that has the

same genetic ma!eup as the

original is calle CLONE.


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History of cloning

" Tapole# $%&'()

" Carp# $%&*+) China - Tong i/hon - 0orl1s first clone fish

" 2ice# $%&3*) - 4irst successfull5 clone mammal - Cha5la!h5an, 6eprence7,

87irio7a, an Ni!itin

" 8heep# $%&&*) - 4rom earl5 em9r5onic cells 95 8teen 0illasen.

4rom ifferentiate em9r5onic cells 95 2egan an 2orag $%&&')

4rom somatic cell - oll5 the sheep $%&&:).

";hesus 2on!e5# Tetra $(eta $2ales, (


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2ichigan 8tate Uni7ersit5 researchers

e7elope a neB, more efficient Ba5of cloning /e9ra fish,

Fe9ra fish is an excellent moel for

unerstaning normal e7elopment

an 9irth efects

Carp Bas the first clone fish, in

China 95 an em9r5ologist Tong

i/hou in %&*+.


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oll5 - 4irst mammal clone

from an ault cell 95 ;oslin

Institute in 8cotlan

8he li7e from %&&* to (


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Types of cloning

2olecular cloning

Cellular cloning

Organism cloning

Create copies of genes or segments of

N=

Create copies of Bhole animals

Create copies of cells


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Molecular cloning

" Arocess of ma!ing multiple copies of a efine N=seuence.

" Use to amplif5 N= fragments containing Bhole genes

" It can also 9e use to amplif5 an5 N= seuence such

as promoters, non-coing seuences an ranoml5fragmente N=.

" It is use in a Bie arra5 of applications ranging from

genetic fingerprinting to large scale protein prouction.


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Recombinant DNA technology or DNA cloning

" = piece of N= from

one organism is

transferre to a self

replicating geneticelement such as a

9acterial plasmi.

" Alasmi is then

transferre to a

9acterium, Bhere

multiple copies of the

same gene are

generate.


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Origin of replication

" To amplif5 an5 N= seuence in a li7ing organism, that

seuence must 9e lin!e to an origin of replication

" Origin of replication is a seuence of N= capa9le of

irecting the propagation of itself an an5 lin!e

seuence.


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Cloning vector

= 7ariet5 of speciali/ecloning vectorsexist.

Cloning 7ector is a

small piece of N=

into Bhich a foreignN= fragments can 9e

inserte.

These 7ectors alloB

protein epression,

tagging, singlestrane ;N= an

N= prouction.


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Cloning of DNA fragment

%. 4ragmentation - 9rea!ing

apart a stran of N=

(. Ligation - gluing together

pieces of N= in a

esire seuence

+. Transfection - insertingthe neBl5 forme pieces

of N= into cells

@. 8creeningselection -

selecting out the cells

that Bere successfull5transfecte Bith the neB

N=


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4ragmentation#

N= of interest nees to 9e

isolate

Ligation

=mplifie fragment is

inserte into a 7ector $piece

of N=).

6ector is linearise using

restriction en!ymes

6ector is incu9ate Bith thefragment uner appropriate

conitions Bith an en/5me,

DNA ligase.


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Transfection

" 6ector Bith the insert of interest is

transfecte into cells.

" Chemical sensiti7ation of cells,

electroporation, optical inection an9iolistics.

Culturing

" Transfecte cells are culture.

Ientification of clones

" Cloning 7ectors inclue selecta9leanti9iotic resistance mar!ers, Bhich alloB

onl5 cells in Bhich the 7ector has 9een

transfecte to groB.

" Cloning 7ectors also contain colour

selection mar!ers, Bhich pro7ie

9lueBhite screening on "#galmeium." 4urther ientification 95 means of AC;,

restriction fragment anal5sis anor N=

seuencing.

Electroporation is atechnique that totemporarily punch holesin cell membranes andferry drugs or genes intothem


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Colour changing gene, lacF

Bas ae

This ena9les the 9acteria to

turn the colourless su9stance

-galIATG 9lue

This gene split apart Bhen the

clone N= is incorporate into

the 7ector

It is possi9le to tell that the

9acteria ha 9een transfecte

$incorporate into the cell) an

also the 9acteria Bas

transfecte Bith the 7ector

containing insert N= or ust

the 7ector alone $remem9ering

that if the 7ector has properl5

incorporate the clone N=, it

Bill ha7e lost its a9ilit5 to

change -gal 9lue)


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Cellular cloning

" Cloning a cell means to eri7e a population of cells from a

single cell.

" In this, a single-cell suspension of cells are expose to amutagenic agent or rug an this is one to ri7e selection

" It is then plate at high ilution to create isolate colonies

" Each arise from a single an potentiall5 clonall5 istinct cell.

" =t an earl5 groBth stage Bhen colonies consist of onl5 a feB ofcells, sterile pol5strene rings $cloning rings), ippe in grease

are place o7er an ini7iual colon5 an a small amount of

tr5psin is ae.

" Clone cells are collecte from insie the ring an transferre

to a neB 7essel for further groBth.


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Cloning cell-line colonies using cloning rings


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History

" 4irst recore cloning 9egan in %&


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$enefits

" There is no nee for an entire creature to 9e su9ecte to

experiments

" 8o meical experiments can 9e one faster, cheaper

an more relia9l5.

" Cellular cloning also eliminates a lot of ethical issues

such as the issue of using animals or human fetuses in

experiments.

" Cellular cloning also has a higher success rate than N=

transference cloning.


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Organism cloning

" Creation of a neB multicellular organism, geneticall5

ientical to another.

" This form of cloning is an asexual metho of reprouction,

Bhere fertili/ation or inter-gamete contact oes not ta!e

place.

ifferent t5pes

" Em9r5o cloning

" ;eproucti7e cloning

" Therapeutic cloning


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%mbryo cloning

" J=rtificial tBinning? or JEm9r5o splittingK.

" 2imics the natural process of creating ientical tBins.

" In nature, tBins occur ust after fertili/ation of an egg cell

95 a sperm cell.

" 0hen the fertili/e egg, calle a /5gote, tries to i7ie into

a tBo-celle em9r5o, the tBo cells separate.

" Each cell continues i7iing on its oBn an ultimatel5

e7elop into a separate ini7iual Bithin the mother.

" 8ince, tBo cells come from the same /5gote, the resulting

ini7iuals are geneticall5 ientical.


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Artificial embryo t&inning

" This occurs in a Aetri ish instea of mother1s 9o5.

" In7ol7es manual separation of ini7iual cells from an earl5em9r5o

" Then alloB each cell to i7ie an e7elop on its oBn Bith the

same N= as the original.

" It is performe at the *- to 3-cell stage, Bhere it can 9e use as

an expansion of 'n#vitro fertili!ation (')*+to increase thenum9er of a7aila9le em9r5os.

" ;esulting em9r5os are then place into a surrogate mother,

Bhere the5 are carrie to term an eli7ere.

" 8ince all the em9r5os came from the same /5gote, the5 gi7e rise

to mono/5gotic $geneticall5 ientical) tBins.

" It has 9een successfull5 carrie out on man5 animals.

" 6er5 limite experimentation has 9een one on human em9r5os.


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Human embryo cloning

" 8tarts Bith a stanar in 7itro fertili/ation

" 8perm an an egg cell are mixe together on a glass ish.

" =fter conception, the /5gote $fertili/e egg) is alloBe to e7elop into

a 9lastula.

" F5gote i7ies first into tBo cells, then four, then eight...

" Chemical is ae to the ish to remo7e the ?zona pellucida?

co7ering." This material pro7ies nutrients to the cells to promote cell i7ision.

" 0ith this co7ering remo7al, 9lastula is i7ie into ini7iual cells

Bhich are eposite on ini7iual ishes.

" The5 are then coate Bith an artificial /ona pellucia an alloBe to

i7ie an e7elop.

" >est results o9taine 95 interrupting /5gote at the tBo cell stage.


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Repro,uctive cloning

" Is to create an animal that has the same N= as another animal.

" oll5 the sheep - first animal create 95 reproucti7e cloning.

" N= from an o7um is remo7e an replace Bith N= from a somaticcell remo7e from an ault animal.

" The fertili/e o7um, calle a pre-em9r5o, is implante in a uterus an

alloBe to e7elop into a neB animal.

" Clones of ault animals are create 95 a process calle

-omatic Cell Nuclear Transfer (-CNT+.

" ;oslin Techniue an Honolulu Techniue.

" ;esulting offspring Bill 9e geneticall5 ientical to the onor an not the

surrogate, unless the onate nucleus is ta!en from a somatic cell of

the surrogate.


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Cloning techni.ues

-omatic Cell Nuclear Transfer

" ;efers to the transfer of the nucleus from a somatic cell to

an egg cell.

" = somatic cell is an5 cell in the 9o5 other than the tBot5pes of reproucti7e cells $germ cells), sperm an egg.

" Example of somatic cells - 9loo cell, heart cell, s!in cell

" E7er5 somatic cell has tBo complete sets of chromosomes,

Bhereas germ cells onl5 ha7e one complete set." Nucleus contains all the information in the form of N= that

cells nee to form an organism.


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" 4irst egg from a female onor is ta!en an its nucleus isremo7e to create an enucleate egg.

" = somatic cell, Bhich contains N=, is ta!en from person

Bho is 9eing clone.

" Then enucleate egg is fuse together." This creates an em9r5o, Bhich 9egins to i7ie normall5.

" Then it is implante into the uterus of the surrogate mother

through in 7itro fertili/ation.

" Clone Bill 9e an exact genetic replica of the ault thatonate the somatic cell nucleus to the egg.


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Difference bet&een fertili!ation an, -CNT

" 4irst ifference lies in those tBo sets are originate.

" In fertili/ation, sperm an egg 9oth contain one set ofchromosomes.

" 0hen the sperm an egg oin, the resulting /5gote ens

up Bith tBo sets - one from the father $sperm) an one

from the mother $egg).

" In 8CNT, the egg cell1s single set of chromosomes is

remo7e.

" It is replace 95 the nucleus from a somatic cell, Bhich

alrea5 contains tBo complete sets of chromosomes.

" The resulting em9r5o contain 9oth sets of chromosomes

come from the somatic cell.


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The Roslin Techni.ue

" ;oslin Techniue is a 7ariation of 8CNT" ;oslin Institute researchers use this to create oll5.

" In this process, somatic cells Bith nuclei intact are

alloBe to groB an i7ie

" The5 are then epri7e of nutrients to inuce the cellsinto a suspene or ormant stage.

" =n egg cell that has ha its nucleus remo7e is then

place in close proximit5 to a somatic cell an 9oth cells

are shoc!e Bith an electrical pulse." Cells fuse an egg is alloBe to e7elop into an em9r5o.

" Then, the em9r5o is then implante into a surrogate.


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The Honolulu Techni.ue

" This techniue Bas e7elope 95 r. Teruhi!o0a!a5ama at the Uni7ersit5 of HaBaii.

" In this metho, the nucleus from a somatic cell is

remo7e an inecte into an egg that has ha its

nucleus remo7e." The egg is 9athe in a chemical solution an culture.

" The e7eloping em9r5o is then implante into a

surrogate an alloBe to e7elop.


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/roblems in repro,uctive cloning

" Clones are not strictl5 ientical since the somatic cellsma5 contain mutations in their nuclear N=.

" =itionall5, the mitochonra in the c5toplasm also

contains N=

" uring 8CNT process, this N= is Bholl5 from the onoregg, thus the mitochonrial genome is not the same as

that of the nucleus onor cell from Bhich it Bas

prouce.

" This ma5 ha7e important implications for cross-speciesnuclear transfer in Bhich nuclear-mitochonrial

incompati9ilities ma5 lea to eath.


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Therapeutic cloning

" Initial stages are ientical to reproucti7e cloning.

" The stem cells are remo7e from the pre-em9r5o Bith the

intent of proucing tissue or a Bhole organ for transplant

9ac! into the person Bho supplie the N=.

" The pre-em9r5o ies in the process.

" The goal of therapeutic cloning is to prouce a health5

cop5 of a sic! person1s tissue or organ for transplant

" 8CNT process use to create em9r5os for therapeutic

purposes.

" This process is also calle ?research cloning?.

" The goal is to har7est stem cells that can 9e use to stu5

human e7elopment an to potentiall5 treat isease.


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" ;esultant em9r5o is alloBe to groB for perhaps %@ a5s.

" 8tem cells Boul then 9e extracte an encourage to groB into apiece of human tissue or a complete human organ for transplant.

" En result Boul not 9e a human 9eing it Boul 9e a replacement

organ, or piece of ner7e tissue, or uantit5 of s!in.

" 4irst successful therapeutic cloning Bas accomplishe in (


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" =ll the information neee to create a neB human 9eing

is containe in each cell of an existing human 9eing." N= testing on a human often starts 95 scraping some

cells from the insie of a person1s mouth.

" Li7ing cells can 9e scrape off of a person1s s!in that

contains the N= of the person" Thus the5 contains all of the information reuire to

prouce a uplicate or clone person.

" Each cell is, in fact, a form of human life for the simple

reason that it contains human N=." = Boman1s o7um also contains her N=.

T ! 1 it N= Thi t it t


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" Ta!e a Boman1s o7um, an remo7e its N=. This con7erts it to a

form of human life.

" ;emo7e the N= from a cell ta!en from a human, an insert it into

the o7um.

" Gi7e an electrical shoc! to the resulting o7um to start up its em9r5oma!ing operation. In a small percentage of cases, a pre-em9r5o Bill

9e forme.

" The pre-em9r5o is alloBe to e7elop an prouce man5 stem cells.

" The proceure is ientical to that use in reproucti7e cloning.

HoBe7er, the pre-em9r5o is not implante in a Boman1s Bom9." -tem cellsare remo7e from the pre-em9r5o this results in its

eath.

" 8tem cells Boul 9e encourage to groB into Bhate7er tissue or

organ is neee to treat the patient.

" 8tem cells are a uniue form of human cell that can theoreticall5

e7elop into man5 organs or 9o5 parts the 9o5.

" Tissue or organ Boul 9e transplante into the patient.


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" Theoreticall5, these stem cells can 9e use to e7elop

into replacement organs $heart, li7er, pancreas, s!in, etc)

" There Bere four main hurles.%. 8tem cells ha7e to 9e ?successfully isolated and grown

in the laboratory.?

(. The5 ha7e to 9e encourage to ?turn into specific cell

types.?+. The5 ha7e to 9e pro7en usa9le in treating patients Bith

iseases, inuries, or isorers.

@. The transplante tissue must e7elop normall5 an

must not represent significant ?risks to the patient.?


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Therapeutic cloning

Clone em9r5o e7elops onl5 to the

9lastoc5st stage. =t this stage, a cluster

of cells M the Inner cell mass $IC2) M is

e7ient at one sie of the em9r5o. IC2 isthe source of pluripotent $totipotent)

em9r5onic stem $E8) cells. These cells

can then 9e inuce to ifferentiate into

specific cell t5pes,These can 9e use to

treat iseases that result from the loss

anor malfunction of particular cells.

;eproucti7e cloning In reproucti7e cloning, after

going through the 2orula stage

an reaching the 9lastoc5st

stage, the clone em9r5o isimplante into the uterus an

alloBe to e7elop further. The aim is for the clone to

e7elop to term an 9e 9orn in

the same Ba5 as non clone

offspring.


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Aluripotent,

em9r5onic stem

cells originate

as inner masscells Bithin a

9lastoc5st.

The stem cells

can 9ecomean5 tissue in the

9o5, excluing

a placenta.

Onl5 the

morula1s cellsare totipotent,

a9le to 9ecome

all tissues an a

placenta.


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A,vantages of Cloning

%. 8olution to infertilit5

(. Aro7ie organs for transplantation

+. Aro7ie treatments for 7ariet5 iseases

@. Genetic moification engineering'. The healthiness of infants

*. >etter unerstaning of genetic iseases

:. Help impro7e li7es

3. Arotects Enangere 8pecies&. Impro7es foo suppl5

Disa,vantages of Cloning


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Disa,vantages of Cloning

01 The %lement of 2ncertainty

>efore the cloning of oll5 Bas achie7e, man5 em9r5os $(::

Nos) Bere estro5e.31 'nheriting ,iseases

Cloning creates a cop5 of the original. = human clone Boul

therefore inherit the genetic traits of its preecessor, Bhich

inclues genetic a9normalities an iseases.

41 The /otential for Abuse

If human cloning 9ecame a realit5 Bhat chec!s an 9alances

Boul 9e put in place to pre7ent a9use 0oul scientists go

o7er9oar Bith the technolog5 If a couple has a clone that the5

are not happ5 Bith, Bhat Boul the5 o next Clones coul 9egroBn in a farm-li!e fashion simpl5 for har7esting organs or

stem cells. The potential for e7aluing human life cannot 9e

ignore.

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Cloning Technique