Title: Making Transgenic Plants and Animals
1Making 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.
- Generate new systems or sources for
bioengineered drugs (e.g., use plants instead
of animals or bacteria).
2Transgenic Mice
The organism of choice for mammalian genetic
engineers. - small - hardy - short life
cycle - genetics possible - many useful
strains and tools
3The 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"
M. Capecchi Univ. of Utah
Sir M. Evans Cardiff Univ., UK
O. Smithies UNC Chapel Hill
4The Problem DNA Integration
- Can occur by homologous (H) or non- homologous
(N-H) recombination - Frequency of N-H gtgt H (by at least 5000-fold) in
mammalian cells - If you want H integrants, which you need for
knock-outs, you must have a selection scheme for
those.
5Vector with a transgene
tk1 tk2 - Herpes Simplex Virus thymidine
kinase genes (make cells susceptible to
gancyclovir) Neo - neomycin resistance
gene Homologous regions - homologous to the
chromosomal target Transgene - foreign gene
6Example of what happens with N-H recombination
Transformed cells are neo-resistant, but
gancyclovir sensitive.
homol--gt
7What 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.
8From Fig. 5.40
- To knock-out a gene
- Insert neo gene into the target gene.
- Transform KO plasmid into embryonic stem cells.
- Perform double-selection to get cells with the
homologous integration (neo gangcyclovir
resistant). - Inject cells with the knocked-out gene into a
blastocyst.
1.
KO
KO
2,3.
9How to make a transgenic mouse.
With DNA
(mouse)
10Chimeric mouse
11- (a) If the recipient stem cells are from a brown
mouse, and the transgenic cells are injected into
a black (female) mouse, chimeras are easily
identified by their Brown/Black phenotype. - (b) To get a completely transgenic KO mouse
(where all cells have KO gene), mate the chimera
with a black mouse. Some of the progeny will be
brown (its dominant), because some of the germ
line cells will be from the KO cells. ½ the brown
mice will have the transgene KO, because the
paternal germ-line cell was probably
heterozygous. - (c) To get a homozygous KO mouse (both
chromosomes have the KO transgene), cross two
brown transgenic heterozygotes. 1/4 will be
homozygous at the transgene locus.
12Not necessarily 31
Similar to Fig. 5.41
13Gene therapy in humans presents some formidable
problems
- If you could introduce the gene in early
development (e.g., eggs? or blastocyst) might
could cure (or partially cure) many diseases. - How to fix them later, as a child, adolescent,
adult, etc.? - Transgenic technology stem cell technology
many interesting possibilities
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