Lecture on transgenic animals.ppt

8/18/2019 Lecture on transgenic animals.ppt

1/25

Transgenic Animals


2/25

A genetically modifed organism (GMO) is anorganism whose genetic material has been alteredusing techniques in genetics, generally known as

recombinant DNA technology

!ecombinant DNA technology is the ability tocombine DNA molecules "rom di#erent sources intothe one molecule

$hus, the abilities or the %henoty%e o" theorganism, or the %roteins it %roduces, can bealtered through the modifcation o" its genes

$ransgenics are genetically modifed organismswith DNA "rom another source inserted into theirgenome

A large number o" transgenic animals ha&e beencreated


3/25

roduction o" $ransgenic Animals

$ransgenic animals can be %roduced by three

di#erent techniques

Method - Microin.ection o" DNA into the%ronucleus o" /ygotes

Method 0 +n.ection o" embryonic stem cellsinto blastocysts

Method 1 2se o" engineered retro&iruses

$he %rocedure is done with the hel% o" acom%lete microin.ection set3containing o" a


4/25

A %ronucleus (%lural %ronuclei) is the nucleus o" as%erm or an egg cell during the %rocess o" "ertili/ation,a"ter the s%erm enters the o&um, but be"ore they "use

http://en.wikipedia.org/wiki/Spermhttp://en.wikipedia.org/wiki/Egg_cellhttp://en.wikipedia.org/wiki/Fertilizationhttp://en.wikipedia.org/wiki/Fertilizationhttp://en.wikipedia.org/wiki/Egg_cellhttp://en.wikipedia.org/wiki/Sperm


5/25

$he %ronuclear microin.ection method o" %roducing

transgenic animals is based on the introduction o" linear DNAsequences into the chromosomes o" "ertili/ed eggs

$he "oreign DNA must be integrated into the genome %rior tothe doubling o" the genetic material that %recedes the frstclea&age in order "or the animal to be born with a co%y o"

this new in"ormation in e&ery cell

*or se&eral hours "ollowing the entry o" the s%erm into theoocyte, the male and the "emale %ronuclei can still be seenindi&idually under a normal light microsco%e and they ha&enot "used yet into a so called /ygote

$he "oreign DNA may be in.ected into either %ronuclei withno di#erence in results4 howe&er, the DNA is ty%icallyin.ected into the male %ronucleus because it is slightly larger and closer to the oocyte sur"ace

$hese oocytes are subsequently trans"erred into the uteruso" seudo3 re nant reci ient animals $he o#s rin is

Method - Microin.ection o" DNA intothe %ronucleus


6/25

• In vitro "ertili/ation is used tostart a transgenic animal

• 5ar&ested eggs and s%erm are"ertili/ed, and be"ore the%ronuclei "use, the transgene isin.ected into the male%ronucleus

• $he embryo continues to di&idein culture and is then im%lantedinto a mouse

• $he 6"oster mother7 mouse hasbeen treated with hormones sothat she acce%ts the embryo

and carries on with the%regnancy

• $he o#s%ring are screened "orstable integration o" thetransgene

'reation o" $ransgenic Animals by Nuclear+n.ection

Gene injected intothe male pronuclei


7/25


8/25

Method 0 +n.ection o" 8 cells intoblastocyst3stage embryos

8mbryonic stem cells (8 cells) are deri&ed "rom the innercell mass o" blastocysts (early embryos) $hese cells are%luri%otent, which means that they can de&elo% into almostany ty%e o" tissue

8 cells are used "or more %recise modifcations o" themouse genome $his technique makes it %ossible to insertas well as remo&e or modi"y DNA sequences 9nock3out,and conditional mutant mice can be %roduced with thismethod

$he frst ste% is the remo&al o" 8 cells "rom a blastocystA"ter trans"ection o" the 8 cells4 selection, cloning andscreening methods make it %ossible to detect 8 cell clonesthat demonstrate the desired, site3s%ecifc recombination

A"ter microin.ection o" the genetically modifed 8 cells intoblastocyst3stage embryos, the 8 cells di&ide and become


9/25

$o create a transgenic animal with embryonic stem cells, thetransgene must frst be inserted into the embryonic stemcells $he stem cells are trans"ormed with the transgene,which integrates by homologous recombination

$hen the stem cells are in.ected into an early male embryo

(blastocyst) o" mouse and the embryo is %ut into a %seudo3%regnant "emale mouse $he o#s%ring are chimeras because


10/25

!etro&iruses can in"ect the cells o" early embryos,including embryonic stem cells 5ence, retro&irus&ectors may introduce transgenes

Disad&antages

•:irus DNA is introduced along with the transgene•!etro&iruses can carry only limited amounts o" DNA•*ounder animals made using retro&iruses arealways chimeras, because insertion o" the &irus doesnot occur e;actly when the nuclei "use

artially transgenic animals (that ha&e some sectorsor tissues altered) are use"ul because the transgenictissues may be com%ared with normal tissue within

the same animal

Method 1 2se o" engineered retro&iruses


11/25

$he frst transgenic animals were mice created by!udol" ?


12/25

Barge $ransgenic Mice illustrate transgenictechnology

'reation o" larger mice by inserting the rat gene "or

growth hormone (omatotro%in)

omatotro%in consists o" a single %oly%e%tide encodedby a single gene omatotro%in gene o" rat was clonedand inserted into "ertili/ed mouse eggs $he transgenic

mice were larger (about twice normal si/e), althoughnot as large as rats

•$he gene was under the control o" the %romoter"rom an unrelated mouse gene, metallothionein ,

which is normally e;%ressed in the li&er

•+nstead o" being made in the %ituitary gland (thenormal site "or growth hormone) the ratsomatotro%in was manu"actured in li&er

5uman somatotro%in has also been e;%ressed in mice


13/25

Barge $ransgenic Mice


14/25

$rendy $ransgenic Mice

Marathon Mouse'an run about -C metersmore than a milebe"oree;haustion $his is twice as "ar as a normal mouse can lastMarathon mouse has enhanced A!3deltaa regulator o"se&eral genes in&ol&ed in burning "at and in musclede&elo%ment

Mighty MouseBack myostatin, a %rotein that slows muscle growth $heresult is colossal muscle de&elo%ment

*ierce Mousehows abnormal aggression Eoth co%ies o" the NR2E1 gene

deleted$he cloned human &ersion o" the NR2E1 gene has been shownto cure the de"ects in brain de&elo%ment and restore normalbeha&ior to ferce mice

mart Mouse

5a&ing im%ro&ed learning and memory5as e;tra co%ies o" the NR2B gene encoding the NMDA


15/25

!ecombinant %rotein %roduction usingtransgenic li&estock roduction o" heterologous %roteins!ecombinant %roteins can be %roduced in the milk "rom

transgenic cows or other "arm animals

$he cloned genes are %laced under the control o" a regulatoryregion that will allow gene e;%ression only in the mammarygland

*or small3scale %roduction, transgenic goats are o"tenused 8g transgenic goats %roducing r$A (recombinant tissue%lasminogen acti&ator)


16/25

9nockout mice "or medical research

$ransgenic animals, mostly mice, are o" great &alue in

the genetic analysis o" inherited diseases and cancer

$he general a%%roach is to inacti&ate, or 6knock out,7 the gene o" interest and then ask what de"ect thiscauses

$he gene is inacti&ated frst and then this co%y is usedto generate knockout

$he incoming DNA, carrying the disru%ted gene,re%laces the original F "unctional co%y o" the gene by

homologous recombination

*ounder mice are obtained that ha&e one co%y o" thedisru%ted gene Ey breeding them together, mice withboth co%ies disru%ted are obtained

+" the gene in question is essential, homo/ygous


17/25

9nockout Mice

$he target gene is cloned anddisru%ted by inserting a DNAcassette

$his work is usually done inbacteria

Once the construct is made,it is %utback into a mouse byin.ection intothe male %ronucleus during

"ertili/ation

A"ter the transgenic o#s%ringare born, two hetero/ygotesare crossed to create a

homo/ygous knockoutmouse


18/25

Bocation e#ects on e;%ression o" the transgene

$ransgenic organisms carrying the same insertedtransgene o"ten di#er considerably in e;%ression

Eoth the le&el o" e;%ression and the %attern o" e;%ressionin &arious tissues o" the body may &ary

Many o" these e#ects are due to the location o" the

transgeneresence o" any nearby regulatory elements like

enhancers$he %hysical state o" the DNA is im%ortant

Determining the %osition e#ect8;tract transgene DNA "rom a transgenic animal inwhich the transgene is not e;%ressed

+nsert the DNA to construct another line o"transgenic animals


19/25

'ombating location e#ects on transgenee;%ression

A Euilding a%%ro%riate regulatory elements in the

transgene construct itsel"E ite3directed insertion

- Dominant control elementsome regulatory sequences control nearby genes or clusters o"

genes in a dominant manner

0 +nsulator sequences or boundary elements$hese sequences block the acti&ity o" other regulatory

elements

+" a gene is anked by two insulator sequences it is %rotected"rom the e#ects o" any regulatory elements beyond theinsulators

$ransgenes anked by insulators %robably "orm inde%endentloo%s o" DNA "romwhich heterochromatin is e;cluded


20/25

rotection o" a Gene by +nsulator equen


21/25

$argeting the transgene to a s%ecifc location

• !equires homologous recombination, as o%%osedto the random integration that usually ha%%enswith in.ected DNA

• +nserting a transgene in a s%ecifc location maybe desirable "or se&eral reasons

- 'hromosomal location o"ten a#ects thee;%ression o" a transgene

0 $he transgene is not necessarily a no&elgene Gene re%lacement in the samelocation, and under the same regulation

• $he DNA to be inserted is anked by sequencesthat are homologous to those at the targetlocation

- +nsertional :ectors0 !e%lacement o" DNA


22/25


23/25

+nducible 8ndogenous romoters

romoter is induced by hea&y metals such as lead, cad

$he heat shock %romoter "rom Drosophila Hsp70 gene

Deliberate control o" transgene e;%ressio


24/25

table e;%ressions o" human %roteins ha&e been

de&elo%ed in many animals, including shee%, %igs, andrats *or e;am%le, 5uman3al%ha3-3antitry%sin, which hasbeen de&elo%ed in shee% and is used in treating humanswith this defciency

$ransgenic %igs with human3histo3com%atibility ha&ebeen studied in the ho%es that the organs will besuitable "or trans%lant with less chances o" re.ection

$ransgenic li&estock ha&e been used as bioreactors

since the -==s Many medicines, including insulin andmany immuni/ations are de&elo%ed in transgenicanimals

+n March 0--, the bioacti&e recombinant 5umanByso/yme was e;%ressed in the milk o" clonedtransgenic cattle

uccess"ul e;am%les o" transgenic animals

http://en.wikipedia.org/wiki/Insulinhttp://en.wikipedia.org/wiki/Immunizationshttp://en.wikipedia.org/wiki/Immunizationshttp://en.wikipedia.org/wiki/Insulin


25/25

+n -===, scientists at the 2ni&ersity o" Guel%h in Ontario,'anada created the genetically engineered 8n&iro%ig

+n 0=, scientists in

Documents

Lecture on transgenic animals.ppt