Transcript Transgenic mice: generation and husbandry

Transgenic


Transgenic: an organism that has had DNA
introduced into one or more of its cells
artificially
“transgenic”: DNA is integrated in a
random fashion by injecting it into the
pronucleus of a fertilized ovum
• Random (approx.. 10% disrupt an endogenous
gene important for normal development)
• multiple copies
Transgenic


Transgenic: an organism that has had DNA
introduced into one or more of its cells
artificially
“transgenic”: DNA is integrated in a
random fashion by injecting it into the
pronucleus of a fertilized ovum
• Random (approx.. 10% disrupt an endogenous
gene important for normal development)
• multiple copies
Transgenic production

Transgenic mice are often generated to
1. characterize the ability of a promoter to
direct tissue-specific gene expression
• e.g. a promoter can be attached to a reporter
gene such as LacZ or GFP
2. examine the effects of overexpressing and
misexpressing endogenous or foreign genes at
specific times and locations in the animals
Brinster's growth hormone mouse
Trangenic mouse embryo in which the promoter for a
gene expressed in neuronal progenitors (neurogenin 1)
drives expression of a beta-galactosidase reporter gene.
Neural structures expressing the reporter transgene are
dark blue-green. (Dr. Anne Calof)
GFP transgenic mouse (Nagy)
9.5 day embryos GFP and wt
Tail tip
GFP transgenic mouse (Nagy)
Injecting fertilized eggs



The eggs are harvested 0.5 dpc
(superovulated or natural matings)
The DNA is usually injected into the male
pronucleus
The eggs can be transferred the same day or
the next (2-cell) into pseudopregnant female
oviducts
Pronuclear injection
Implantation of 1 or 2 cell
embryos


The injected eggs are implanted the same
day or are incubated overnight and
implanted the next day
Injected eggs are transferred to the oviduct
of a 0.5 dpc pseudopregnant female
Implanting 1(or 2) cell embryos
1
2
Implanting 1(or 2) cell embryos
(cont.)
3
Pseudopregnant females and
vasectomized males




Female mice can be tricked into thinking
they are pregnant
A mouse in estrus is mated with a
vasectomized male
pseudopregnancy
If eggs (blastocysts) implanted will become
truly pregnant and will give birth to live
offspring
Vasectomizing
1
2
Breeding Tg founders


Individually backcrossed to the strain of
choice
DO NOT intercross different founders each founder results from a separate
RANDOM transgene integration even
Transgenic mice as tools

Study gene function
• Many human diseases can be modeled by
introducing the same mutation into the mouse.
Intact organism provides a more complete and
physiologically relevant picture of a transgene's
function than in vitro testing

Drug testing
Transgenic mice as tools



Polio virus receptor
Normal mice can't be infected with polio
virus. They lack the cell-surface molecule
that, in humans, serves as the receptor for
the virus.
Tg mice expressing the human gene for the
receptor can be infected by polio virus and
even develop paralysis and other
pathological changes characteristic of the
disease in humans