Mutational analysis of loxP sites for efficient Cre‐mediated insertion into genomic DNA

Abstract

Summary: The Cre/loxP system has been used in transgenic models primarily to excise DNA flanked by loxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant loxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing λ integrase, mutant loxP sites were placed at the E. coli attB site and the excision-insertion ratios of incoming DNA plasmids carrying a second, complementary mutant loxP site were determined. Comparison of 50 mutant loxP sites combinations to the native loxP 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 loxP combinations resulted in 1,421-fold and 1,529-fold increases in relative integration rates over wild-type loxP sites. These loxP mutants could be exploited for site-directed “tag and insert” recombination experiments. genesis 36:162–167, 2003.