Mutational analysis of LoxP sites for efficient Cre-mediated insertion into genomic DNA

The Cre/IoxP system has been used in transgenic models primarily to excise DNA flanked by IoxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant IoxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing lambda integrase, mutant IoxP sites were placed at the E. coli attB site and the excision-insertion ratios of incoming DNA plasmids carrying a second, complementary mutant IoxP site were determined. Comparison of 50 mutant IoxP sites combinations to the native IoxP site revealed that mutations to the inner 6 bp of the Cre binding domain severely inhibited recombination, while those in the outer 8 bps were more tolerated. The most efficient IoxP combinations resulted in 1,421-fold and 1,529-fold increases in relative integration rates over wild-type IoxP sites. These IoxP mutants could be exploited for site-directed tag and insert recombination experiments. genesis 36:162-167, 2003. (C) 2003 Wiley-Liss, Inc.