Rv. Merrihew et al., EFFICIENT MODIFICATION OF THE APRT GENE BY FLP FRT SITE-SPECIFIC TARGETING/, Somatic cell and molecular genetics, 21(5), 1995, pp. 299-307
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.