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|ES-cell gene targeting
(Knock-out or knock-in)
|ES-cell gene targeting
Information from non-Cancer Center principal investigators must be on file before you use shared resources facilities. (See registration information.) For more information on any service, click the name of the service.
A desired DNA construct is injected into the pronucleus of a 0.5 d.p.c. Embryo. Resulting pups are returned to the Client. The client will then analyze these by Southern blot to determine the presence of the given transgene.
Mouse Embryonic Stem cells that have undergone homologous recombination are injected into the blastocoel cavity of 3.5 d.p.c. blastocyst stage C57Bl6 Embryos. Injected blastocysts are implanted into pseudopregnant recipient females, and chimeric pups are born. Chimerics are returned to the Client. Those that transmit the 'knock-out' allele to germline produce agouti pups, which are analyzed for phenotype by the client.
A targeting vector containing a 'knock-out' or 'knock-in' gene of interest is electroporated into Mouse Embryonic Stem Cells (129 SvJ). The cells undergo drug selection, clones are picked and expanded in tissue culture, then returned to the Client for analysis. Homologous Recombinant clones are verified by the client using either PCR or Southern Blot analysis by the client.
Chromosome counting is done on targeted homologous recombinant ES cell clones prior to blastocyst injection for quality control.
Embryo Rederivation is used to create a pathogen free strain of mice from lines that arrive at the University from a non-approved vendor and mice contaminated with various pathogens (MHV, Pinworm, Parvovirus). Males arrive in the Rodent Isolation Unit (RIU), and wild-type females of the desired background strain are superovulated and mated to the males. Embryos are harvested in the RIU, washed and transferred into pathogen free, pseudopregnant recipient females, which are returned to the Client.
Sexually mature females that are infertile or cannot carry a litter to term are ovariectomized. These ovaries are then transplanted into the wild-type ovariectomized females of a given background strain, and immediately returned to the Client. Two weeks later, the recipients are mated by the client to produce transgenic pups of the original genotype.
Genetic banking through the use of mouse embryo cryopreservation is a practical means to store scientifically valuable mice. Mouse embryos stored in liquid nitrogen offer a safe way to preserve lines with potential future use but no current use and offer savings in facility space and the expense associated with keeping a breeding colony. Another advantage is that during the process of embryo collection, most pathogenic organisms are excluded as in other methods used to rederive lines of mice.
Briefly, the procedure entails harvesting embryos from donor females at the 8-cell developmental stage that is optimal for cryopreservation by our method. A controlled-rate methanol bath freezer is used to gradually cool the embryos to -80°C, before plunging them into liquid nitrogen for storage. Quality control is assured by freezing and thawing a representative sample of wild-type frozen embryos along with each line frozen down. Frozen embryos should be retrieved by the investigator one week post-freezing for long-term storage in liquid nitrogen in the investigator's own lab.