What is a transgenic animal?• An animal contains a foreign gene (genes)introduced purposely by human intervention•Transgenic animals are altered so that their DNAproduce proteins that normally they would notproduce
History of transgenic animal production 1970s, first transgenic mice via viral infection, but not germline transmission 1980s, first transgenic mice via microinjection, the most popular technique 1985, first transgenic rabbits, sheep, pigs and cattle 80-90, commercial transgenic services, via transgenic facility 1990s, transgenic farm animal companies as bioreactors and organ donors
Different ways to create transgenic animalsTarget gene – transgene Promoter/enhancer - when, where, how much Coding sequence - coding the specific protein ploy A detail - mRNA stability Transgene Structure
Gene transfer methods1. Microinjection of recombinant DNA into the male pronucleus of an in vitro fertilized egg.2. Embryonic stem cell transfer (ES).Other methods: 1. Chemical or 2. Viral delivery into ES cells, or homologous recombination with ES cells.
Microinjection• Inject DNA molecules (transgenes) directly intomale pronucleus• Most popular technology, commercial available• Success rates range from 10-30% depending onskills and constructs•Efficiency is not related to the copies of transgenesinjected
Microinjection• The technique can be applied to other species• No theoretical limit for the size of the construct• Overall efficiency is still low, particularly for farmanimals• Tandem repeat of gene constructs (head-tail)• High frequency of mosaic• Initial investment is high
Virus mediated gene transfer• Earliest method for successful gene transfer inmammals• Virus has transfection property• Killed virus is replication defective• The virus gene is replaced with transgene gene• The transgene is delivered to the host cell bytransfection (gene therapy)• Can be used to transfect a wide range of cells, e.g.,ES cells
Virus mediated gene transfer•Direct transfection of embryos has resultednongermline transgenics• ES cells transfection has resulted in germlinetransgenics• Has succeeded in chickens and fish• Transfecting oocytes resulted in 100% transgenics• Only small transgene construct is usable (8 kb orless)• More research is needed on the safety of themethod
Embryonic stem cells•Used mostly when trying to target a transgene toa specific site in the genome.• Derived from ICM of blastocyst stage embryo• Divide in vitro indefinitely without differentiation• Contribute to development of the fetus in anytissues, organs (germline)• Has the potential to give rise to all tissues
ES cells• May be transfected with transgene or withgenes removed (knockout) or inserted prior tomicroinjection• Has revolutionized genetics, development,immunology and cancer research in mice
Steps for ES Cell Manipulation.
Approaches to using ES cells to create transgenic animals. 1. The transgene can be microinjected into the ES cells 2. can be introduced by a virus, 3. Chemical (e.g calcium phosphate or rubidium chloride 4. by using homologous recombination.
Nuclear transfer• Creation of Dolly• Somatic cells be transfected, or genetically alteredprior to NT• 100% efficiency of any progeny• Low efficiency• Abnormal development
Screening for Transgenic Positives• Identification of transgene integration - DNA• Detect transgene transcription - mRNA• Detect transgene expression - protein
TRANSGENIC ANIMAL CLASSIFICATION
Five major categories: 1. disease models 2. transpharmers 3. xenoplanters 4. food sources 5. scientific models1.Disease models: animals that have been modified to exhibit the symptoms and progression of a particular disease, so that treatments for that disease can be tested on them (e.g oncomouse, AIDS mouse etc)
2.Transpharmers: animals modified to express a particular protein or suite of proteins in their milk to avoid animal sacrifice when obtaining the drug. The proteins can be purified to produce medicines and hormones to treat humans, or can possibly be administered as medicinal milk itself.
Mice- commonly used to test the transpharmingtransgene first.The transgenic procedure is promising, but veryexpensive, and still has a low success rate especiallyfor larger farm animals. A mouse engineered in 1987 to express the clot dissolver drug tissue plasminogen activator (tPA) In 1990, human alphaantitrypsin, an inhibitor used to treat emphysema, was produced in the mouse’s milk. 1997 at the New Technology Institute- human alpha- lactalbumin in mouse’s milk
Larger animals like sheep, goats, and cows are the targetsfor large-scale transpharming. E.g 6 transgenic lambs for Roslin Institute - created in 1997 to produce a human clotting factor in their milk. The first transpharmer goats were produced in 1991 at the Tufts University School of Veterinary Medicine to produce tissue plasminogen activator, a clotdissolving drug. transpharmer goats were produced in 1999 using SCNT contained high levels of human antithrombin III.
The first transgenic cow (Gen Pharm Intern, California),dubbed “Herman”, and his first transgenic offspring werebred at Gen Pharm’s lab (Netherlands)Two calves were produced by microinjection of DNA intoembryos that were then implanted in surrogate mothers andborn alive.One of these cows was female the transgenerearranged itself so that a portion of the lactoferrin cDNA wasdeleted.The other calf was male, later called “Herman.” He and hisoffspring contained the correctly arranged gene for humanlactoferrin.
3. Xenoplanters: animals that have been engineered to not express the foreign antigens that normally prevent the transplantation of their organs into humans.4. Food sources: animals that grow bigger or faster to produce more food in a shorter amount of time with fewer resources. E.g superpig, superfish
5. Scientific models: animals producing more or less of a particular protein than usual, Study that protein’s purpose in biological mechanisms or development applied to humans. E.g ANDi first transgenic monkey
TRANSGENIC ETHICS1. Animal Rights Versus Animal Welfare2. Right to meddle in the genomes of living beings Transgenesis- a logical step beyond selective breeding, open doors past what we previously have known to cure diseases!!?? possibly end world hunger entirely!!!??Transgenic Art - Creating monsters!!! E.g “Alba,” therabbit that glows under UV light!!!!???
Eduardo “transgenic art.” refers to animals and plants with aplanned genome intended to express an artistic ideasymbolized by the proteins they code for.
4. Animal Death Versus Human Lives Saved low success rate in creating transgenic animals.5. Transgenic Animals and the Environment decrease of genetic variability within that species Transgenic animals are not “more fit” than their “normal” cousins. 6. Transgenic Oversight Transgenic experimentation should be as humane as possible. 7. Religions and Transgenic Ethics
Applications of transgenic farm animals• Agricultural applications• Bioreactors• Organ/cell/tissue donors• Basic research/disease model