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类似LOX的序列。这些异源特异的lox位点还允许Cre重组酶利用Cre/loxP整合重组系统将外源Ψ有效地整合到宿主基因组中自然产生的内源性伪IoxP(LoxP)序列中。我们研究了Cre/ΨloxP系统是否有助于转基因的定点整合和提高转基因动物的生产效率。这是首次报道Cre介导的整合性重组靶向于小鼠早期胚胎中被称为伪Loxm5(ΨLoxm5)的内脏吸虫Loxm5序列。首先，我们在胚胎环境中鉴定了Cre/Ψloxm5系统。将Cre表达载体与位于两个假loxm5位点两侧的跨克隆(CMV/LacZ基因)共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，并对重组产物进行调查。结果表明，在小鼠胚胎环境中，Ψbloxm5位点是Cre介导的重组的活性底物。转基因效率高达27%(6/22)。转基因幼鼠体内的ΨLoxm5位点与转基因基因发生了定点整合。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样位点的有效且简单的策略。

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.