EFFICIENT MODIFICATION OF THE APRT GENE BY FLP FRT SITE-SPECIFIC TARGETING/

The FLP/FRT site-specific recombination system was established and cha racterized at the APRT gene in CHO cells. Targeting frequencies with F LP-stimulation were about 1 to 5 x 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT(+) ce ll lines were analyzed by Southern blotting 56% were FLP-targeted inte grants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal si te-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologo us recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate ch romosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegit imate recombination.