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

在酵母菌和哺乳动物基因组中已经鉴定出功能性的lox样序列。这些异特异性的lox位点也允许Cre重组酶利用Cre/loxP整合重组系统特异性地将外源DNA整合到宿主基因中自然存在的内源性伪ioxp （ΨLoxP）序列中。我们研究了Cre/ΨloxP系统是否有助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。这是首次报道cre介导的针对早期小鼠胚胎中内源性lox样序列（称为伪loxm5 （ΨLoxm5））的整合重组。首先，我们描述了胚胎环境中的Cre/Ψloxm5系统。将Cre表达质粒和一个带有两个伪loxm5位点的跨清（CMV/LacZ基因）共注射到受精卵母细胞原核中。将注射的卵子移植到养母体内，并研究重组产物。结果表明Ψbloxm5位点在小鼠胚胎环境中是Cre介导重组的活性底物。转基因效率达27%（6/22）。在转基因幼崽中发现了转基因基因与内源性ΨLoxm5位点的位点特异性整合。我们的研究结果表明，Cre/Ψloxm5整合重组系统是一种针对哺乳动物胚胎内源性lox样位点的有效而简单的策略。

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.