This deals with transgenesis, history of transgenic animal, methodology , some examples of transgenic animals, importance, advantage and disadvantage of transgenic animals
2. TRANSGENESIS
• Transgenesis : Phenomenon of introduction of exogenous
DNA into genome to create and maintain stable heritable
characters.
• Transgene : Foreign DNA which is introduced.
• Transgenic animal: One whose genome is altered in
heritable manner by foreign DNA sequence introduction
into genome of its zygote or embryo(GEO/GMO) with
new genetic character.
• Transgenesis is facilitated by liposomes,enzymes,plasmid
vectors, viral vectors, pronuclear injection, protoplast
fusion and DNA injection.
HISTORY
• 1973: First GMO was created by S.Cohen and H.Boyer.
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3. • 1974: First transgenic animals mice by Rudolf Jaenisch (SV40
viral DNA inserted in a mouse genome).
• 1980: First transgenic mice via microinjection.
• 1982: worlds first expressing transgenic animal (Super mouse-
inserting a human growth hormone gene in mouse genome)-
offspring will be larger than parents.
• 1985: First transgenic rabbits,sheep,pigs and cattle.
• 1996- 5-July: DOLLY was female domestic sheep and first
mammal cloned from an adult somatic cell by nuclear transfer
– Sir lan Wilmut , Keith Campbell and colleagues.
PRODUCTION/METHODOLOGY
• STEP 1: Construction of transgene –made of three parts:
Promoter, Gene to be expressed and Termination sequence.
• STEP 2: Introduction of foreign gene into animal.
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4. • STEP 3: Screening of transgenic positives: Progeny are screened
by PCR to examine site of incorporation of gene.
• Some will not express as it integrated into transcriptionally
inactive site.
• STEP 4: Further animal breeding is done to obtain maxima
expression-Heterozygous offsprings are mated to form
homozygous strains.
RETROVIRAL VECTOR METHOD
• Transfer of small piece of DNA
carried by retroviruses.
• Results in chimera-org consists
of tissues or parts of diverse
genetic constitution.
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5. MICROINJECTION
METHOD
•Super ovulation of young virgin female
mice of 4-5 weeks by administration of
follicle stimulating hormone followed by
HCG(2 days).
•Superovulated mouse produces 30-35
eggs (not normal 5-10 eggs).
•Female mice are mated with males and
fertilized eggs are removed from
fallopian tubes.
•By micromanipulation using a
microinjection needle & holding pipette
DNA is injected into male pronucleus.
•Eggs with transgenes are kept overnight
in incubator to develop two cell stage.
•Eggs are implanted microsurgically to
pesudopregnant mother which is mated
previous night with vasectimized male.
•Foster mother delivers pups after 3
weeks of implantation.
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6. EMBRYONIC
STEMCELL
METHOD
•Cells are taken from inner cell mass
of blastocyst of developing embryo-
proliferate in cell culture-pleuripotent
ES cells.
•It involves-introduction of foreign
DNA into ES cells. And this in culture
allows for gene manipulation using
electroporation/microinjection.
•Desired GE cells –identified by
selection process using marker gene
(thymidinekinase,enzymes-
dihydrofolate reductase and neomycin
phosphotransferase) and PCR.
•Transfected cells cultured-introduced
into blastocyst and then implanted into
pesudopregnant female mouse.
•Transgenic founder mice is produced.
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7. EXAMPLES OF TRANSGENIC ANIMALS
• TRANSGENIC MICE: Used as animals models for
study of human diseases and for production of therapeutic
agents.
HUMAN MOUSE: Transgenic mice with human immune
system produced-human mice.
• Choose Mice with SCID and human thymus from aborted
fetus–transplanted under capsule membrane of mouse kidney.
• Human lymph node on opposite kidney of mouse. After week,
immature immune cells from human fetus injected into mouse
tail vein.
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8. • These lymphocytes enters thymus under
kidney and mature to T- lymphocytes.
• This will enters circulation and lymph node.
• Multiply to form full-pledged functional
immune system. After 2 weeks ,Transplant
mice display human immune system.
APPLICATION:
• Boon for immunologists for working on AIDS
for possible development of AIDS vaccine .
ALZHEIMER’S MOUSE:
• Transgenic mice were developed by
introducing amyloid precursor gene into fertilized
egg cells of mice.
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9. • Synthesis of human amyloid protein and plaques
accumulation in mice brain.
APPLICATION:
• Understanding pathological basis of disease.
ONCOMOUSE:
• I-developed for breast cancer.
• Introduction of chimeric DNA that contains c-myc gene &
sections of MMT virus into fertilized egg cells of mouse.
• Breast cancer in adult female mice and trait passed to
offspring.
APPLICATION:
• Understanding of cancer and cancer therapy.
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10. KNOCKOUT MOUSE FOR ALLERGY:
• Developed by removing gene encoding for receptor protein so
Ab cannot binds to cells due to lack of receptors and it is
unaffected by allergic reactions.
KNOCKOUT MOUSE WITH MEMORY LOSS AND
RETINITIS PIGMENTOSA:
• Develops mice with lack of hippocampus so that it lacks
ability to remember.
• Inactivation of mouse rhodopsin gene, rod cells in retina gets
degenerated which is comparable to human retinitis
pigmentosa.
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11. TRANSGENIC CATTLE
• Transgenic cow – over expressed casein
transgene – milk with high content of casein.
• Lactase transgene is introduced and expressed
in mammary gland- milk free from lactose will
be secreted.
• Creation of transgenic cattle with improved
Resistance to viral,bacterial and parasitic
Diseases.
TRANSGENIC SHEEP WITH INCREASED
WOOL PRODUCTION:
• Development of transgenic sheep has bacterial
genes for synthesis of cysteine.
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12. • Two enzymes synthesized by transgenes –traps H2S liberated
in intestine to produce cysteine.
TRANSGENIC PIGS:
• It can produce human haemoglobin and separated by pig
haemoglobin by AT.
• Used for organ transplantation by harvesting human organs in
it.
• Because physiology of pig is close to humans, so organ
transplant from transgenic pigs into human showed successful
result.
TRANSGENIC FISH:
• SUPER FISH
• Developed with increasing in their growth & size.
• Introduction of growth hormone transgene by microinjection
method.
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13. • Fertilized eggs with inserted transgene
are incubated in temperature regulated tanks.
• Transgenic salmon fish is 10-12 times
larger than normal fishes.
GLO FISH:
• Genetically modified zebra fish ,produce by
integrating FP gene from jelly fish into fish
embryo.
DOLLY: First mammal clone developed by
Wilmut & Campbell in 1997.
• Sheep with mother and no father.
TRANSGENIC MOSQUITOES:
• Genes coding for endotoxin of P.vivax
Introduced into mosquito zygote.
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14. • Produces toxin of malarial parasites in their saliva and inject
toxin to human blood through sucking blood.
• Human immune system produce Ab against toxin and resist
malarial parasites.
TRANSGENIC MONKEY AND RABBIT:
• ANDi –first transgenic monkey in 2000, inserted DNA spelled
backward and insertion of GFP into its genome.
• Proves transgenic primates can be created and express foreign
gene delivered into their genome.
• ALBA, inserted with enhanced GFP in 2000.
IMPORTANCE OF TRANSGENIC ANIMALS:
• Medical-disease model,pharma use, organ transplantation.
• Agricultural- disease resistant animals, improving quality and
quantity of milk,egg,wool production, breeding.
• Industrial-toxicity sensitive transgenic animals to test
chemicals. Produce proteins and enzymes for industrial
reaction.
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15. • ADVANTAGES:
• Increased growth rate.
• Improved disease resistance.
• Increased muscle mass.
• Improved nutritional quality.
• Improved wool quality.
• DISADVANTAGE:
• Inserted gene has multiple functions.
• Breeding issues.
• Mutagenesis
• Low survival rate.
RECENT TRENDS:
• Transgenic goats to produce human breast milk.
• Bioluminescent transgenic mouse for study of mammary
gland tumor development.
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