Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (=lox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/=lox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (=loxm5) in early mouse embryos. We characterized the Cre/=loxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by =loxm5 and =loxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the =loxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous =loxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/=loxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.
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