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Making Transgenic Plants and Animals

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hardy - short life cycle - genetics possible - many useful strains and tools ... UNC Chapel Hill. The Problem: DNA Integration ... – PowerPoint PPT presentation

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Title: Making Transgenic Plants and Animals


1
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.
  • Generate new systems or sources for
    bioengineered drugs (e.g., use plants instead
    of animals or bacteria).

2
Transgenic Mice
The organism of choice for mammalian genetic
engineers. - small - hardy - short life
cycle - genetics possible - many useful
strains and tools
3
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"
M. Capecchi Univ. of Utah
Sir M. Evans Cardiff Univ., UK
O. Smithies UNC Chapel Hill
4
The 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.

5
Vector 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
6
Example of what happens with N-H recombination
Transformed cells are neo-resistant, but
gancyclovir sensitive.
homol--gt
7
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.
8
From 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.
9
How to make a transgenic mouse.
With DNA
(mouse)
10
Chimeric 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.

12
Not necessarily 31
Similar to Fig. 5.41
13
Gene 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

users.rcn.com/jkimball.ma.ultranet/BiologyPages/
T/TransgenicAnimals.html
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