Development and use of production method for transgenic animals using Cre/pseudo IoxP system

在酵母和哺乳动物基因组中已经鉴定出功能性lox样序列。这些异源特异性lox位点还允许Cre重组酶使用Cre/loxP整合重组系统特异性靶向外源DNA到内源性假IoxP（pseudo-IoxP）序列中的有效整合，所述内源性假IoxP（pseudo-IoxP）序列天然存在于宿主基因组中。我们研究了Cre/CryloxP系统是否可用于转基因的位点特异性整合和提高转基因动物的生产效率。这是第一次报告Cre介导的整合重组靶向内分泌lox样序列称为假loxm 5（pseudo-loxm 5）在早期小鼠胚胎。首先，我们在胚胎环境中的Cre/Cryloxm 5系统的特点。将Cre表达质粒和两侧带有两个假loxm 5位点的translene（CMV/LacZ基因）共显微注射到受精小鼠卵母细胞的原核中。将注射的卵子转移到养母体内并研究重组产物。结果表明，Cre Bloxm 5位点是Cre介导的重组在小鼠胚胎环境中的活性底物。转基因效率达27%（6/22）。在转基因幼鼠中发现转基因位点特异性整合到内源性p53 Loxm 5位点中。我们的研究结果表明，Cre/Crloxm 5整合重组系统是一个有效的和简单的策略，在哺乳动物胚胎中的内源性lox样位点的目标。

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-IoxP (ΨLoxP) sequences that occur naturally in the host genorne using the Cre/loxP integrative recombination system. We investigated whether the Cre/ΨloxP system is useful for site-specific integration of a transgene and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endocienaus lox-like sequence termed pseudo-loxm5 (ΨLoxm5 ) in early mouse embryos. First we characterized the Cre/Ψloxm5 system in the embryonic environment. Cre expressing plasmid and a transclene (CMV/LacZ gene) flanked by two pseudo loxm5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers and recombination products were investigated. The results show that the Ψbloxm5 site is an active substrate far 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 ΨLoxm5 site was found in the transgenic pups. Our findings demonstrated thai the Cre/Ψloxm5 integrative recombination system was an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.