Making Transgenic Plants and Animals. Why? Study gene function and regulation Generate new organismic tools for other fields of research. Cure genetic diseases. Improve agriculture and related raw materials.
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The organism of choice for mammalian genetic engineers.
- short life cycle
- genetics possible
- many useful strains and tools
The Nobel Prize in Physiology or Medicine, 2007
Mario R. Capecchi, Martin J. Evans and Oliver Smithies
for their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells"
Univ. of Utah
Sir M. Evans
Cardiff Univ., UK
UNC Chapel Hill
tk1 & tk2 - two copies of a Herpes Simplex Virus thymidine kinase gene (makes cells susceptible to gancyclovir)
Neo- neomycin resistance gene
Homologous regions - homologous to the chromosomal target
Transgene - foreign gene
Example of what happens with N-H recombination
Transformed cells are neo-resistant, but gancyclovir sensitive.
What happens with HR
If DNA goes in by HR, transformed cells are both neo-resistant and gancyclovir-resistant!
Use double-selection to get only those cells with a homologous integration event.
From Fig. 5.40
How to make a transgenic mouse.
(a) If the recipient ES cells are from a brown mouse, and the transformed (transgenic) ES cells are injected into a black (female) mouse, chimeras are easily identified by their Brown/Black phenotype.
(b) To obtain a completely transgenic KO mouse (where all cells have a KO gene), mate the chimera with a black mouse. Some of the progeny will be brown (which is dominant), indicating fertilization with a germ-line cell (gamete) that ultimately came from a KO-ES cell. Only about 50% of the brown progeny mice, however, will have the KO allele, because the transgenic ES cell that underwent meiosis to produce the germ-line cell was probably heterozygous for the KOed gene.
(c) To obtain a homozygous KO mouse (both alleles are KOs), cross brown heterozygotes, and ~1/4 of the progeny will be homozygous.
Not necessarily 3:1
Similar to Fig. 5.41