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Gene-Targeting is the intentional modification of an animal's genome in a very precise process utilizing homologous recombination. The technology to produce gene-targeted animals is currently a moving target. Historically, the vast majority of gene-targeted animals have been made via the electroporation of traditional plasmid-based vectors into rodent Embryonic Stem cells (ESC). Typically, the targeting vector contains a selection cassette to identify the ES cell colonies that had stably incorporated the vector. These colonies are then cloned and the clones are screened for homologous recombination using PCR and Southern blots. The gene-targeting frequency can vary wildly and can even vary between different ESC clones from the same strain of mouse. The range is 0% to 90%. Many things affect gene-targeting efficiencies. Perhaps one of the most important is the locus to be modified. Another factor is the type of modification requested. We have found that very large deletion tend to occur at low frequencies even in a locus that was amenable to alternative types of gene-targeting such as a knockin of a single base pair polymorphism. This facility offers this service. What is required to get started is a suitable gene-targeting vector and a Completed Gene Targeting Service Request form (link).
Please be advised that the Knockout Mouse Program (KOMP) repository at
UC Davis and the International Gene-Targeting Consortium (IGTC)
repository have produced ESC clones for a majority of the known mouse gens. The IGTC clones are often gene knock out first and can be
converted to conditional gene knockouts by the expression of a
site-specific recombinase (Flipe). The ES cell clones from these
repositories are very reasonably priced and will save time and money if
they are used.
This facility eventually will be able to design and construct all manner gene-targeting vectors. Presently, we offer the electroporation of mouse ESC with investigator provided gene-targeting vectors. We also provide detailed vector design and construction consultation at no charge.
The most onerous element of gene-targeting is not the identification of gene-targeted ES cell clones. It is the breeding of coat color chimeras to identify those mice that pass the gene-targeted mutation through the germ line. There are breeding schemes that can accelerate this process however there is no identified marker that says that an ES cell clone will go through the germ line. This facility only uses ESCs that are proved germ line competent. However, there is only one analysis that has been reported to be a100% reliable means of predicting germ line transmission (GLT). That is to run PCR on mating plugs from chimera matings. Typically, only male mice are bred to establish GLT. Male mice that produce targeted allele positive plugs will produce targeted allele carrying founders sooner or later.
A new method that the this facility is eager to test is the direct creation of gene-targeted mice via the TALEN methodology.This methodology uses uniquely designed site-specific endonucleases to stimulate homologous recombination with a gene-targeting vector. The great promise of this technology is that it produces founders directly without going through the intermediate of chimeric mice.
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