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Background  Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of  lox  sites with mutations in the LE or RE of the  lox  sequence has previously been developed. Recombination between LE and RE mutant  lox  produces a wild-type  lox P site as well as an LE+RE double mutant  lox  site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using  lox 71 (an LE mutant) and  lox 66 (an RE mutant) in ES cells. Recently, other LE/RE mutant  lox  sites showing higher recombination efficiency in  Escherichia coli  have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.    Results  Using ES cells, we compared six RE mutant  lox  sites, focusing on their recombination efficiency with  lox 71. All of the RE mutant  lox  sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant  lox  site, under continuous and strong Cre activity in ES cells. Two RE mutants,  lox JTZ17 and  lox KR3, produced more stable LE+RE double mutant  lox  than did the  lox 66/71 double mutant.    Conclusion  The two mutant RE  lox  sites,  lox JTZ17 and  lox KR3, are more suitable than  lox 66 for Cre-mediated integration or inversion in ES cells.

Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells