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Transgenic Animals
V SUSHMA
10Y71S0114
M.Pharmacy
Dep.of Pharmacology
Samskruthi college of
Pharmacy Dep. of Pharmacology
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Introduction
Transgenic animals:
◦ Animals which have been genetically engineered to containone or more genes from an exogenous source.
◦ Transgenes are integrated into the genome.
◦ Transgenes can be transmitted through the germline toprogeny.
◦ First transgenic animal produced = “Founder Animal”
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Transgenic Animals: Definition
A transgenic animal is one that carriesa foreign gene that has beendeliberately inserted into its genome.
The foreign gene is constructedusing recombinant DNA methodology .
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Why are (they)transgenic animals beingmade
Reasons for producing transgenicanimals:
Obtain fundamental information on
living organisms and human diseases. Use animals as a source of
pharmaceutical proteins
Improve animal production
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Historical background :
Historical background
In 1977 Gurdon transferred mRNAand DNA into Xenopus eggs.
In 1980 Brinster et al. used fertilizedmouse ova Gordon and Ruddle.
1981 coined the term “transgenic “
animals carrying new genes
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EXAMPLES OF TRANSGENICANIMALS
Rabbit
Sheep
Goats
Cow Pigs Chicken
Fish
fly
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Transgenic animals-Advantages
Increased growth rate
Improved disease resistanceimproved foodconversion rates
Leaner meat Increased muscle mass
Improved nutritionalquality
Improved wool quality
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Production of transgenicanimals1. Plan the experiment.
2. Transgene DNA preparation.
3. Insertion of the transgene into animals.
4. Selection of gene targeted cells.
5. Clone and verify the integrity of the transgene..
6. DNA purification.
7. Establish a screening method.
8. Establish an expression assay.9. Screen potential founders
10. Breeding and analysis of transgenic rodents
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Production of transgenic animal(overview)
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Transgenic Animals: Methods
Classical
- Pronuclear Microinjection
- Lentiviral Infection
- Embryonic Stem (ES) Cell Gene Transfer
- ES Cell mediated Gene Targeting (knock-out,knock-in)
Experimental
- Transfection of Somatic Cells - Cloning
- Sperm Based Transfection (ICSI)
- Transposons
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Samskruthi
college of Pharmacy Dep. of Pharmacology
Why the Mouse?
• The closest to humans – mammal
• The most complex -integration of systems (endocrine, immune, nervous etc.)
Of the model organisms which may be geneticallymodified, the mouse is:
• Genetic manipulation is extremely versatile – Gain-of-Function (Transgenesis), Loss-of-Function(knock-out), Change-of-Function (knock-in);temporally and spatially restricted (conditional)
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Example of transgenic mouse
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Mouse Transgenesis Methods
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ooo
Pronuclear microinjection
Lentivral infection
ES based transgenesis
pros
Relatively simple and efficient
Long transgenes possiblePotentially all species
Very efficientSingle copy insertions
No technical equipmentWorks in many species
Long transgenes possibleGene targeting possibleSingle copy insertions
cons
Random integration
Multicopy insertions( Strain limitations)
High embryo mortality9.5 kb packaging limit
Safety issues (?)Only random integration
Technically difficultTime consuming
Species / Strain limitations
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PharmacyDep.of Pharmacology
Pronuclear Microinjection
• Microinjection of DNA directly into the pronuclei of fertilized eggs
• Implantation of the microinjected eggs into a surrogate mother
• Allowing the embryos to develop to birth
• Demonstrating that the foreign gene has been stably incorporated intothe host genome and that it is heritable in at least one of the offspring
• Demonstrating that the gene is expressed and regulated correctly inthe host organism
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Institute of Laboratory
Animal Science University of Zurich
Transgene DNA
Analyse the progenyby PCR or Southern blotting
Production of transgenic miceby pronuclear microinjection
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Microinjection station
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Institute of Laboratory
Animal Science University of Zurich
Production of transgenic mice
by ES cell gene transfer
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C57BL/6Blastocyst
(black)
ES cells129/SvJ(agouti)
agouti black
ES cell injection into blastocyst
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Germline male chimera (C57BL/6 in BALB/c)with offspring
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Timeline: Transgenesis by PronuclearMicroinjection or Lentiviral transfection
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Matefounders
0 2 4 8 10 126
DNA or LVinjection
gestation
maturationof founders
gestation
Birth
Identyfyfounders
maturationof F1 progeny
Beginanalysis
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Timeline: generation of ES cell-derived mice
Samskruthi College of
Pharmacy Dep. Of Pharmacology
Introducetargetingvector intoES cells
Identifyhomologousrecombinantsby DNAanalysis
Identify mouseChimeras withhigh ES cellcontribution Germline transmission
Beginanalysis
0 2 4 8 10 126
Drugselection Colony growthand expansion Injectclonesintoblastocysts
Sexualmaturationof chimeras
Identifymale andfemaleheterozygotes
Sexualmaturation ofheterozygotes
Identifyhomozygotes
gestation gestation
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Economic Traits:
Scientists created cattle to producemilk containing certain human proteinswhich may help in the treatment of
emphysema. It also helps in breeding. Farmers can
produce transgenic animals that have
the needed traits for doing a specific job.
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Medical Research:
Every year people die because of lackof a replacement heart, liver, orkidney.
Transgenic pigs may be able to supplythese organs.
Research is trying to find a way to
remove a pig protein and replace itwith a human protein
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Samskruthi college of
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Trends in the Field of Transgenic Animals
• More refined transgene systems:
- temporal regulation (tet ON/OFF)
- tissue specific regulation ( Cre/lox)
- RNAi induced gene silencing- YAC/BAC transgenesis
- dominant negative transgenes
• Integrative Databases• Animal Welfare Aspects
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Transgenic Animals: PotentialProblems
Technical problems to closely mimic a
desired situation Underestimation of biological
complexity
Mouse – Human differences
Inappropriate analysis
Undefined genetic backgrounds
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THANK YOU
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