Abstract
Gene targeting has become an extremely popular method for generating mouse models of human disease. The targeting event is carried out in vitro in pluripotent embryonic stem (ES) cells, which are then used to generate chimeric founder animals by either fusion with early-stage embryos or direct injection into donor blastocysts. Strategies used range from the simple insertion of vector sequences into the chosen gene or replacement of crucial exons with vector sequence, to complicated spatial or temporal knockouts and chromosomal rearrangements. All these methods depend on homologous recombination between the endogenous ES cell genomic DNA and the targeting construct, which must share a minimum stretch of DNA sequence. Consequently, they all require extensive preliminary physical mapping and restriction mapping before designing a construct for use in the targeting event. This is followed by subcloning of the desired restriction fragments into a vector backbone.
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