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

为了通过显微注射将转基因整合到小鼠基因组中的特定位点，一个有效的位点特异性重组系统是必不可少的。我们开发了Cre突变的LOX系统，以促进Cre重组酶的整合反应。我们使用了异源lox位点，这些位点不会与野生型loxP重新结合，而是本身。比较了两个lox序列lox511和lox2272在ES细胞中的重组效率。建立了3株携带lox71-BSR基因的细胞系-lox511-lox2272，并通过电位法引入了Cre表达载体和替换载体lox66-lacZ-lox511和/或lox2272。由于lox71、loxS11和lox2272分别只与lox66、lox511和Iox2272重组，因此通过Cre介导的重组将ES基因组上的bsr基因替换为lacZ基因。我们发现lox66/71和lox2272组合的效果最好。然后，我们尝试通过显微注射的方法建立Cre介导的受精卵定点整合系统。转基因CAG启动子-EGFP-lox71-puro-pa-lox511-lox2272获得转基因小鼠。将Cre表达载体和替换载体lox66-IRES-NLSLacZ-lox511-lox2272显微注射到受精卵的原核中。我们检测了85个转基因阳性胚胎，但没有检测到定点整合事件。由于转基因小鼠携带了多个拷贝的转基因，接下来，我们用携带lox71和lox2272的陷阱载体进行基因捕获获得了三个小鼠系，并携带了单拷贝的陷阱载体PU-17，剪接受体-lox71-Bgeo-loxP-pa-lox2272。用Cre表达载体和含有lox66-EGFP-pa-lox2272的替换载体注射了约2000个卵子，但只有一个胚胎发生了靶向替换。我们现在正在努力寻找显微注射的最佳条件，以提高效率。

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.