Attempts to integrate a transgene into a specific locus in the mouse genome by microinjection to fertilized eggs

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In order to integrate a transgene into a specific locus in the mouse genome by microinjection, an efficient site-specific recombination system is indispensable. We have developed Cre-mutated lox system to promote integrative reaction by Cre recombinase. We used heterospesific lox sites that do not recombine with the wild type loxP but itself. Two lox sequences, lox511 and lox2272, were compared with their recombination efficiencies in ES cells. Three cell lines carrying a construct of lox71-bsr gene-lox511-lox2272 were established, and introduced the Cre-expression vector and the replacement vector, which is lox66-LacZ-lox511 and/or lox2272, by electoporation. Since lox71, loxSll and lox2272 are recombined only with lox66, lox511 and Iox2272 respectively, thebsr gene on the ES genome is replaced by the LacZ gene through Cre-mediated recombination. We found that the combination of lox66/71 and lox2272 gave the best efficiency. Then, we tried to establish Cre-mediated site-directed integration system by microinjection into fertilized eggs. Transgenic mouse lines were produced with the transgene of CAG promoter-EGFP-lox71-Puro-pA-lox511-lox2272. The Cre expression vector and the replacement vector, which is lox66-IRES-NLSLacZ-lox511-lox2272, were microinjected into the pronuclei of the fertilized eggs. We examined 85 transgene positive embryos, but no site-directed integration event was detected. Since the transgenic mice carried several copies of the transgene, next, we used three mouse lines which were obtained by gene trapping with the trap vector carrying lox71 and lox2272 and carry single copy of the trap vector pU-17, splice acceptor-lox71-bgeo-loxP-pA-lox2272. About 2000 eggs were injected with the Cre expression vector and the replacement vector containing lox66-EGFP-pA-lox2272, however, only one embryo showed targeted replacement. We are now trying to find optimal conditions of microinjection to improve the efficiency.