Confirmation of recombination site functionality in gene targeting vectors using recombinase-expressing bacteria.

Recognition sequences for the site-specific DNA recombinases Cre and FLP are commonly incorporated into gene targeting vectors for the purposes of removing selection markers or generating conditional alleles. Gene targeting vectors typically contain a positive selection marker, such as the neomycin resistance gene, flanked by loxP sites. Thus, the selection marker can be removed by breeding to a mouse strain which expresses Cre recombinase in its germ line. Conditional knockout vectors typically have one or more exons flanked by loxP sites and the positive selection marker flanked by FRT sites. Thus, the selection marker is removed with FLP recombinase and the knockout allele is generated in tissues expressing Cre recombinase. Because the generation of mice by gene targeting in embryonic stem (ES) cells is an expensive and time-consuming process, it is important to confirm that the recombination sites in your targeting vector are functional prior to electroporation of ES cells. This chapter describes a simple method for testing the functionality of loxP and FRT sites in vivo using Cre- or FLP-expressing bacteria.