Transgenic an organism that has had DNA introduced

Transgenic: an organism that has had DNA introduced into one or more of its cells artificially w “transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum w • Random (approx. . 10% disrupt an endogenous gene important for normal development) • multiple copies

Transgenic: an organism that has had DNA introduced into one or more of its cells artificially w “transgenic”: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum w • Random (approx. . 10% disrupt an endogenous gene important for normal development) • multiple copies

Transgenic production w 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 Lac. Z 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) w The DNA is usually injected into the male pronucleus w The eggs can be transferred the same day or the next (2 -cell) into pseudopregnant female oviducts w

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 w Injected eggs are transferred to the oviduct of a 0. 5 dpc pseudopregnant female w

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 w A mouse in estrus is mated with a vasectomized male w pseudopregnancy w If eggs (blastocysts) implanted will become truly pregnant and will give birth to live offspring w

Vasectomizing 1 2

Breeding Tg founders Individually backcrossed to the strain of choice w DO NOT intercross different founders each founder results from a separate RANDOM transgene integration even w

Transgenic mice as tools w 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 w Drug testing

Transgenic mice as tools Polio virus receptor w 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. w 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 w