Topic 7: Transgenic Animals and Reproductive Biotechnology

Transgenesis

The process of introducing a foreign gene (transgene) into an animal.

Founder

The first transgenic animal generated from a successful gene transfer procedure.

Germline Integration

The requirement that a transgene be incorporated into the ovaries or testes to be passed on to future generations.

Pluripotent (Totipotent) Cells

Unspecialised stem cells, such as those from a blastula, capable of differentiating into any specialised functional cell type.

Pronuclear Injection

The preferred method for mice and cows where DNA is injected into the male pronucleus of a fertilized egg before fusion.

ES Cell Transfection

Transgene introduction into cultured embryonic stem cells which are then microinjected back into a blastocyst; highly successful in creating knockout mice.

Retroviral Gene Transfer

Using viral vectors to infect embryos; limited by a small DNA capacity (8kb) and the risk of random integration into oncogenes.

Homologous Recombination

A precise process where a defective DNA construct replaces a functional existing gene at a specific chromosomal location.

Positive Selection (neo^r)

The use of the Neomycin phosphotransferase gene in a vector to ensure only transformed cells survive when treated with antibiotics.

Negative Selection (tk)

Using the viral thymidine kinase gene to kill cells where random integration occurred; tk converts Ganciclovir into a toxin.

Chimeric Mouse

A founder animal produced from ES cell transfer that contains a mix of host cells (e.g., black) and modified donor cells (e.g., agouti/brown).

Molecular Pharming

The use of transgenic animals as "bioreactors" to produce human pharmaceuticals (like Factor VIII or insulin) in milk or eggs.

Lysostaphin Gene

A transgene expressed in cow milk to kill Staphylococcus aureus, providing resistance to mastitis and lowering production costs.

Recombinant Bovine Somatotrophin (rBST)

A growth hormone produced in E. coli that, when injected into cows, increases milk production by 25%.

Enviropig

A transgenic pig expressing the E. coli phytase gene in its saliva, reducing faecal phosphorus by 75% to prevent water pollution. *Pigs are unable to digest phytase.

Glo-fish

Zebra fish with fluorescent proteins used as a bioassay; the metallothionein promoter induces fluorescence in the presence of heavy metals.

Antifreeze Proteins

Genes from the Winter Flounder that, if introduced to other fish like salmon, could extend their temperature range for survival.

Mosaicism

A problem in microinjection where not all cells in the developing embryo successfully integrate the transgene.

Transgenic Bird Challenges

Difficulty identifying the male pronucleus due to multiple sperm entry and the tough membrane/shell surrounding the egg.

Aging and Telomerase

Handwritten note: Chromosomes shorten with every division (loss of telomeres); telomerase adds "junk" to ends to protect useful genes, but activity declines with age.

Nuclear Cloning (SCNT)

The process of transplanting nuclei from somatic cells into enucleated eggs to produce genetically identical copies of superior animals.

Transformation vs Transfection

Transformation refers to introducing foreign DNA into prokaryotic cells; Transfection refers to eukaryotic cells.

Superovulation

Females are injected with Pregnant Mares Serum (PMS), followed by human Chorionic Gonadotrophin (hCG) 48 hours later to increase egg production from ~5 to 35.

Verfication of Transgene Presence

Established using Southern blot or PCR analysis to check for the DNA sequence and its appropriate expression.

Ganciclovir Mechanism

A nucleotide analog that the viral thymidine kinase (tk) gene converts into a toxin to kill any cells where random integration occurred.

Mammary Specific Promoter Example

β-casein is a promoter sequence used to ensure human pharmaceutical proteins are expressed specifically in milk.

How are knockout mice produced?

1. Vector Construction: A plasmid is designed with a defective version of the target gene, including a positive selection marker (neor for neomycin resistance) and a negative selection marker (tk for ganciclovir sensitivity).

2. ES Cell Selection: Embryonic stem (ES) cells are transfected. Through homologous recombination, the defective gene replaces the functional one. Cells are treated with Neomycin (to kill non-transformed cells) and Ganciclovir (to kill cells with random integration where the tk gene was not lost).

3. Chimera Creation: Selected ES cells (e.g., from an agouti mouse) are microinjected into a host blastocyst (e.g., from a black mouse) and implanted into a surrogate.

4. Breeding: Resulting chimeras are mated to produce heterozygous (+/ko) lines to ensure the transgene is in the germline. Finally, two heterozygotes are crossed to produce homozygous (ko/ko) knockout mice

Problems with Microinjection Method

1. Stage at which embryo transferred to surrogate
2. Opacity of embryo cytoplasm of some species obscures nuclei
3 Surviving injection
4. Not all embryos or cells have DNA integrated
5. Random insertion can disrupt genefunction - gene therapy
7. Low or no expression

Transgenic Cow

Most common method is microinjection.
➢ Eggs collected from the slaughterhouse
➢ Mature eggs in vitro
➢ Fertilised in vitro
➢ Centrifuge eggs to clear yolk and see pronuclei
➢ Microinjection male pronucleus
➢ Develop to blastula stage
➢ Screen blastula cells for transgene
➢ Implant
➢ Birth