Transgenic animals Hoza, A.

S BLS 209 Lecture

What is a transgenic animal? An animal contains a foreign gene (genes) introduced purposely by human intervention Transgenic animals are altered so that their DNA produce proteins that normally they would not produce

History of transgenic animal production 1970's, first transgenic mice via viral infection, but not germline transmission

1980's, 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 1990's, transgenic farm animal companies as bioreactors and organ donors

Different ways to create transgenic animals

Target gene transgene Promoter/enhancer - when, where, how much

Coding sequence - coding the specific protein

ploy A detail - mRNA stability

Transgene Structure

Gene transfer methods

1. 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 into male pronucleus

Most popular technology, commercial available

Success rates range from 10-30% depending on skills and constructs Efficiency is not related to the copies of transgenes injected

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 farm animals 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 in mammals 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 by transfection (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 resulted nongermline transgenics ES cells transfection has resulted in germline transgenics Has succeeded in chickens and fish Transfecting oocytes resulted in 100% transgenics

Only small transgene construct is usable (8 kb or less) More research is needed on the safety of the method

Embryonic stem cells Used mostly when trying to target a transgene to a 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 any tissues, organs (germline) Has the potential to give rise to all tissues

ES cells

May be transfected with transgene or with genes removed (knockout) or inserted prior to microinjection
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 altered prior 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 models

1.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 transpharming transgene first. The transgenic procedure is promising, but very expensive, and still has a low success rate especially for 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 mouses milk. 1997 at the New Technology Institute- human alphalactalbumin in mouses milk

Larger animals like sheep, goats, and cows are the targets for 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 were bred at Gen Pharms lab (Netherlands) Two calves were produced by microinjection of DNA into embryos that were then implanted in surrogate mothers and born alive. One of these cows was female the transgene rearranged itself so that a portion of the lactoferrin cDNA was deleted. The other calf was male, later called Herman. He and his offspring contained the correctly arranged gene for human lactoferrin.

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 proteins purpose in biological mechanisms or development applied to humans. E.g ANDi first transgenic monkey

TRANSGENIC ETHICS 1. Animal Rights Versus Animal Welfare 2. 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, the rabbit that glows under UV light!!!!???

Eduardo transgenic art. refers to animals and plants with a planned genome intended to express an artistic idea symbolized 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