Transgenic Animals: The ability to manipulate the genome of the whole animal and the production of transgenic animals has influenced the science dramatically in the last 15 years..pdf

Transgenic Animals
Since the early 1980s, fruit flies, fish, sea urchins, frogs, laboratory mice and farm animals, such as cows, pigs,
and sheep have been successfully produced.
The ability to manipulate the genome of the whole animal
and the production of transgenic animals has influenced the science dramatically in the last 15 years. The procedure for introducing exogenous donor DNA into
a recipient cell is called Transfection. Chromosomes are taken up inefficiently so that intact chromosomes rarely survived the procedure. Instead the recipient cell usually get a part of the DNA.

1. Animal Biotechnology
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2. Transgenic Animals
Since the early 1980s, fruit flies, fish, sea urchins, frogs,
laboratory mice and farm animals, such as cows, pigs,
and sheep have been successfully produced.
The ability to manipulate the genome of the whole animal
and the production of transgenic animals has influenced
the science dramatically in the last 15 years.
The procedure for introducing exogenous donor DNA into
a recipient cell is called Transfection.
Chromosomes are taken up inefficientlyso that intact
chromosomes rarely survived the procedure. Instead the
recipient cell usually get a part of the DNA.
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3. Ananimalthatgainsnewgeneticinformationfromtheadditionof
foreignDNAisdescribedasTransgenicwhiletheintroducedDNA
iscalledthetransgene.
Thetransgenesareintroducedintothepronucleioffertilizedeggs
byinjection,andtheinjectedembryosareincubatedinvitroor
implantedintotheuterusofapseudopregnantfemalefor
subsequentdevelopment.
WhatisPronucleus?Forashorttimeafterfertilization,themale
pronucleusandfemalepronucleusexistseparately.
Femalepronucleus;Inthematuringoftheovumpreparatoryto
impregnation,apartofthegerminalvesiclebecomesconverted
intoanumberofsmallvesicles,whichaggregatethemselvesintoa
singleclearnucleuswhichtravelstowardsthecenteroftheeggand
iscalledthefemalepronucleus.
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4. Male pronucleus; In impregnation, the
spermatozon which enters the egg soon
loses its tail, while the head forms a nucleus,
called the male pronucleus, which gradually
travels towards the female pronucleus and
eventually fuses with it, forming the first
segmentation nucleus. The male pronucleus
is larger than the female’s and can be seen
fairly easily under a light microscope.
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5. 5

6. Transgenesis process;
Plasmids carrying the gene of interest are injected into
the germinal vesicle (nucleus) of the oocyte or into the
pronucleus(before uniting with the gamete) of the
fertilized egg.
The egg is implanted into a pseudopregnantmouse
After birth, the recipient mouse can be examined to see
whether it has gained the foreign DNA and if so whether
it is expressed.
As a result; multiple copies of transgenes are integrated
at random locations in the genome of the transgenic
individuals.
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7. Transgenesis;Methodology
Transgenictechnologyhasbeendevelopedand
perfectedinthelaboratorymouse.Sincethe
early1980’shundredsofdifferentgeneshavebeen
introducedintovariousmousestrains.Thesestudies
havecontributedto;
understandingofgeneregulation
tumordevelopment,exampleintroducingoncogenes
andobservetheeffect
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8. immunological specificity, example producing knockout genes that
are responsible for some immunological aspects
molecular genetics of development
other biological interests such as examining the possibility of
using transgenic animals in the industrial production of human
therapeutic drugs.. etc.
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9. Methodsofgenetransferinanimals
Fortransgenesis,DNAcanbeintroducedintomiceby
oneofthefollowingmethods;
Retroviralvectorsthatinfectsthecellsofanearlystage
embryopriortoimplantationintoareceptivefemale.
Microinjectionintotheenlargedspermnucleus(the
malepronucleus)ofafertilizedegg
Introductionofgeneticallyengineeredembryonicstem
cellsintoanearlystagedevelopingembryopriorto
implantationintoareceptivefemale.
Transferofdiploidsomaticnucleiintoanenucleated
oocyte.
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10. Retrovirus-MediatedGeneTransfer
Retrovirusescanbeusedforthetransferof
foreigngenesintoanimalgenomes.
Thiscanbestbedoneat4-16cellstage
embryos,butwithincompleteinfectionsi.elow
infectivityrate
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11. Immediatelyfollowinginfection,theretrovirusproduces
aDNAcopyofitsRNAgenomeusingitsreverse
transcriptase.
Completionofthisprocessrequiresthatthehostcell
undergoestheSphaseofthecellcycle.Therefore,
retroviruseseffectivelytransduceonlymitoticallyactive
cells.
Modificationstotheretrovirusfrequentlyconsistof
removalofstructuralgenes.
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12. TheDNAcopyoftheviralgenome,orprovirus,
integratesrandomlyintothehostcellgenome,usually
withoutdeletionsorrearrangements.
Veryhighratesofgenetransferareachievedwiththe
useofretroviruses.
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13. Disadvantagesofthismethodinclude:
Lowcopynumberintegration.
Additionalstepsrequiredtoproduce
retroviruses.
LimitationsonthesizeoftheforeignDNAinsert
(usually9to15kb)transferred.
Potentialforundesiredgeneticrecombination
thatmayaltertheretrovirus.
Possibleinterferencebyintegratedretroviral
sequencesintransgeneexpression.
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14. Thegenomeoftheretroviralstraincanbeintegrated
intothesamenucleusasthetransgene.Thismeans
thatthevirusitselfcouldbeproducedbythe
transgenicorganismandcreateaproblemespecially
iftheanimalwillbeusedforproductionoffood.
Alsotheprovirusattractsmethylationwhichpossibly
inconjugationwithothermechanismsdisablesits
expressionwhenitpassesthroughthegermline.
Duetothis,andtotheavailabilityofotheralternative
methods,theretroviralvectormethodisrarelyused
forproducingtransgenicanimalsthathavea
commercialpotential.
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15. DNA Microinjection Method
Becauseofthedisadvantagesoftheretroviralvectors,microinjection
ofDNAiscurrentlythepreferredmethodforproducingtransgenic
mice.
First-youneedthegeneofinterestintheproperform.Alinear
transgeneconstructismade,whichcontains:
thestructuralgeneofinterest,withintrons
astrongmousegenepromoterandenhancertoallowthegenetobe
expressed
vectorDNAtoenablethetransgenetobeinsertedintohostDNA
Theimmaturefemalemicewillbeinducedtosuperovulateby
sequentialadministrationandmatedtofertilemales.One-celled
embryosareflushedfromtheoviductsandplacedinadropof
mediumandviewedbyphase-contrastorinterferencemicroscopy.
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16. Thisprocedurehasthefollowingsteps;
Thenumberofavailablefertilizedeggsthat
aretobeinoculatedareincreasedby
stimulatingdonorfemalestosuperovulate.
Thiscanbedoneby
Givingthemiceaninitialinjectionofpregnant
mare’s(anadultfemaleofhorseorrelated
mammal)serum
Anotherinjectionabout48hourslaterofhuman
chorionicgonadotropin(hCG).Bythisprotocolthe
femaleproducesabout35eggsinsteadofthe
normalnumberof5-10.
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17. These females are mated, then sacrificed and the fertilized eggs
(oocytes) are flushed from their oviducts and recovered.
Eggs are treated with hyaluronidase to remove adherent follicle
cells.
Unfertilized eggs are discarded
The eggs are inoculated immediately with the transgene, briefly;
embryo at the pronuclear stage is held in place by suction.
a micro needle loaded with a suspension of plasmid DNA will be
prepared.
It is introduced through the plasma membrane into the most accessible
pronucleus (usually the male) and
several hundred molecules of the recombinant DNA are injected in a
volume of approximately 1 picoliter (p1).
on a good day several hundred eggs can be injected.
The male pronucleus can be located by using dissecting
microscope and the eggs then can be maneuvered, oriented and
held in place while the DNA is microinjected.
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18. 18
oocyte
Micro needle
Pippet

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20. Gene Transfer to Animal cells

21. There are four major strategies for gene transfer
to animal cells, two of which are considered
biological mechanismsusing virus (transduction)
or bacterial that invade animal cells
(bactofection), those methods involve infection
while the other two are chemical and physical
methodswhich do not involve infection thus
termed transfection.
Virus (transduction); the transferred gene
represents part of the viral genome
Bacteria (bactofection); the gene will be
transferred as a plasmid

22. Chemical transfection
 ;DNAwillbetakenfromthesurroundingswhentheDNAispresented
asasyntheticcomplexeitheras;
acomplexwithoverallpositivecharge,allowingittointeractwith
negativelychargecellmembrane andpromoteuptakeby
endocytosis
aslipophiliccomplexthatfuseswiththecellmembrane and
depositsthetransgenedirectlyintothecytoplasm.

23. Physical transfection;
in this method, naked DNA is deposited
directly into the cell by exploiting a physical
force. This includes;
microinjection
particle bombardment
ultrasound
electroporation
Which ever method used, the result is called
transformationwhich is a change in the
recipient cell’s genome caused by the
acquired transgene.

24. Chemical Transfection Techniques
Calciumphosphatemethod;involvesthe
formationofafineDNA/calciumphosphate
co-precipitatewhichfirstsettlesonthecells
andtheninternalizedbyendocytosis.
Theprecipitatemustbeformedfreshlyat
thetimeoftransfection.TheDNAescapes
andreachesthenucleusandcanbethen
expressed.Sincethecellsmustbecoated
bythecalciumcomplex,monolayersofcells
mustbeusedformaximum efficiency.
However,thismethodgivesonly1-2%
transfectionefficiency.

25. Physical transfection methods
•Electroporation
•Ultrasound
both methods create transient pores in the cell

26. Electroporation
isaphysicaltransfectiontechniqueinvolves
creatingtransientnano-metersizeporesin
thecellmembranebyexposingcellstoa
briefpulseofelectricity.Themostcritical
parameteristheintensityanddurationof
electricalpulse.
Electroporationcanbeusedforinvivogene
transferparticularlyforsurfaceornear
surfacetissuesuchasskin,muscleand
certaintumorsoreveninternaltissuessuch
asliver.Thiscanbeachievedbydirect
applicationofelectrodestotheskin
followingshavingandmildabrasionwiththe
DNAbeinginjectedintotheskinbefore
electroporation.

27. Ultrasound transfection
Involves the exposure of cells to a rapidly oscillating
probe such as the tip of sonicator. In this method the
application of ultrasound waves to a dish or cells or a
particular tissue results in the formation and collapse
of bubbles in the liquid, including the cell membrane,
a process known as cavitation.
The transient appearance of such cavities allows the
DNA to cross the membrane into the cytoplasm. This
method can be used both for in vivoor in vitroas the
plasmid DNA is left structurally intact. As for
electroporation, the DNA will be injected and then
the ultrasound will be applied.

28. DNA microinjection
DirecttransferofDNAintothecellwithout
acarrieriscalledDNAmicroinjectin.This
canonlybedoneforonlyfewcellsata
time.Thistechniqueisusedmainlyfor
largecellssuchasoocytes,eggsandthe
cellsofearlyembryos.TheDNAcanbe
directlyinjectedintotissues,suchasskin,
muscleorinternalorgansoritcanbe
injectedintotheblood.

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Theprocessisremarkablyefficient.Upto60-66%
oftheembryossurviveinjectionandupto25-30%
oftheembryostransferredtotheoviductsurviveto
birthandabout25%ofpupsaretransgenic
(transgenicfounders).Thus,from1000inoculated
fertileeggs,30-50(3-5%)transgenicpupsare
produced.
TheinjectedDNAgetsincorporatedatrandom
siteswithinthegenomeandoftenmultiplecopies
areincorporatedatonesite,therefore,notallthe
transgenicanimalswillhavethedesiredtraits.

30. Particle bombardment
isanotherdirectdeliverymethodinitiallydevelopedforthe
transformationofplants.Thismethodinvolvescoatingsmallmetal
particleswithDNAandacceleratingthemintotargettissuesusinga
powerfulforcesuchastheblastofhighpressuregasoranelectric
dischargethroughawaterdroplet.Inanimals,thismethodisusedfor
tissuessuchasskincellsinvivoratherthanculturedcells.

31. Bacterial vectors for gene transfer
Theexploitationoflivingbacteriaforgene
transferiscentraltothegeneticmanipulation
ofplants.Agrobacteriumtumefaciensandits
closerelativeshavebeenusedfor20yearsto
generatetransgenicplants.
Recently,Agrobacterium tumefacienshas
beenusedtotransferDNAtohumancells.

32. How does the bacterial transfer of DNA
happen?
The bacteria invades the host animal cells and
undergo lysis within them releasing their
plasmid DNA. Example of these bacteria
including Salmonellaspecies (lysis occurs in the
phagocytic vesicle), Listeria monocytogenes
and Shigella flexneri(lysis occurs for these two
species after they escape from the vesicle).

33. TheplasmidDNAthenfindsitswaytothe
nucleuswhereitgetsincorporatedwiththe
cell’sgenomeandgetsexpressed.

34. Viruses That are used as gene transfer vectors
Virusparticleshaveanaturalabilitytoadsorb
tothesurfaceofthecellsandgainentry.This
canbeexploitedtodeliverrecombinant
DNAintoanimalcells.
Severalclassesofviruseshasbeenusedfor
genetherapyandatleast8hasbeenusedin
clinicaltrials.Transgenes may be
incorporatedintoviralvectorseitherby
additiontothewholegenome orby
replacingoneormoreviralgenes.Thiscanbe
donebyligationorbyhomologous
recombination.

35. Ifthetransgenereplacesanoneessential
genethevectorisdescribedashelper-
independent
Ifitreplacesanindispensablegene,thenthis
vectorwillbehelperdependent.
Itisgenerallyrecommended tousevectors
fromwhichallviralcodingsequenceshas
beendeletedsuchvectorsaredescribedas
fullydeletedorguttedorgutlessvectors.These
vectorscontainjustthecis-actingelements
requiredforpackagingandforgenome
replication.

36. Advantages of such vectors
high capacity for foreign DNA
because no viral gene products are made, the
vector has no intrinsic cytotoxic effects.
Adeno virus
These are viruses with a linear, double stranded
genome, of approximately 36 kb. These are used
frequently due to certain advantages;
stability
high capacity for foreign DNA
wide host range including none-dividing cells
Ability to produce high titer stocks up to 10
11
pfu/ml.

37. Adeno-associate virus (AAV)
Thesevirusesarenotgeneticallyrelatedto
adenovirusbutisso-calledbecauseitwasfirst
discoveredasacontaminantinanadenovirus
isolate.
TheAAVisasinglestrandedDNAandisa
memberoftheparvovirusfamily.
Itisnaturallyreplicatingdeficient,thusit
requiresthepresenceofanothervirussuchas
adenovirustocompleteitsinfectioncycle.

38. ThedependenceofAAVonaheterologous
helpervirussuchasadenovirusprovidesan
unusualdegreeofcontrolovervector
replicationmakingAAVoneofthesafest
vectorstouseforgenetherapy.
Otheradvantagesofthisviralvectoristhe
widehostrangethatitexhibitsincluding
nonedividingcells.

39. Baculovirus vectors (BV)
Baculoviruspromote high level of transgene
expression in insect cells but can also infect
mammalian cells.
Have rode shape capsid and large double-
stranded DNA genomes. They productively
infect arthropods particularly insects.
BV vectors are used mainly for high–level
transient protein expression in insects and
insect cells.