Making Transgenic Plants and
Animals
• Why?
1. Study gene function and regulation
2. Generate new organismic tools for other
fields of research.
3. Cure genetic diseases.
4. Improve agriculture and related raw
materials.
5. Generate new systems or sources for
bioengineered drugs (e.g., use plants
instead of animals or bacteria).
The organism of choice for mammalian
genetic engineers.
- small
- hardy
- 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"
M. Capecchi
Univ. of Utah
Sir M. Evans
Cardiff Univ., UK
O. Smithies
UNC Chapel Hill
The Problem with trying to make
KOs: Random DNA Integration
• DNA can integrate into the genome by
homologous (H) or non-homologous
(N-H) recombination
• Frequency of N-H >> 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.
Vector for integrating a transgene by HR
(i.e., into a specific site)
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
Nonhom ologous re com bination
homologous
tk1 sequence
neo
transgene
homologous
sequence
tk2
chromosom e
tk1
homologous
sequence
neo
transgene
homologous
sequence
tk2
chromosom e
Transformed cells are neo-resistant, but gancyclovir sensitive.
homol-->
What happens with HR
Hom ologous re com binants
homologous
tk1
sequence
neo
transgene
homologous
sequence
tk2
chromosom e
homologous
sequence
neo
transgene
homologous
sequence
chromosom e
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
To knock-out a
gene:
1. Insert neo gene
into the target
gene.
2. Transform KO
plasmid into
embryonic stem
cells.
3. Perform doubleselection to get
cells with the
homologous
integration (neo &
gangcyclovir
resistant).
4. Inject cells with
the knocked-out
gene into a
blastocyst.
1.
KO
KO
2,3.
How to make a transgenic mouse.
Transfection
With DNA
Blastocyst
Embryonic
Stem
cells
(ES cells)
(mouse)
Grow in culture.
Select for those that carry the transgene.
Inj ect into a blastocyst
Inject into a blastocyst
Implant int o
pseudo pregn an t
mou se
Chimeric 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
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