表达Cre重组酶Cre-pCEP4载体的构建及其在Cre/loxP重组系统中的应用

Construction of Cre recombinase expression vector Cre-pCEP4 and its application in Cre/loxP system

目的:构建表达Cre重组酶的载体Cre-pCEP4,并验证其能够有效识别loxP位点,为人类疾病动物模型的建立提供依据。方法:构建重组载体Cre-pCEP4和pStop-eGFP,利用Fugene HD转染猪胚胎成纤维细胞(PEF)和MCF-7细胞系,利用荧光显微镜观察绿色荧光蛋白表达情况。结果:成功构建重组载体Cre-pCEP4和pStop-eGFP,将2个载体瞬时共转染PEF;经潮酶素B筛选出Cre重组酶稳定表达的MCF-7细胞系瞬时转染pStop-eGFP,在荧光显微镜下观察2种细胞均有绿色荧光蛋白的表达。而单独转染pStop-eGFP的MCF-7细胞系和PEF均未见绿色荧光蛋白的表达。结论:重组载体Cre-pCEP4在细胞内能够表达Cre重组酶,并且表达的Cre重组酶能够识别loxP位点,删除两同向loxP间的DNA片段。

Objective To construct the Cre rebombinase expression vector and identify its function of recognizingloxP sites and provide the basis for establish ment of the ani mal models of hu man diseases . Methods Therecombinant vectors Cre-pCEP4 and pStop-eGFP were constructed . The MCF-7 cells and porcine e mbryonicfibroblasts（PEF）were transfected by Fugene HD. The expression of eGFP was analyzed by fluorescencemicroscope . Results The recombinant vectors Cre-pCEP4 and pStop-eGFP were constructed successfully and theywere co-transfected into PEF . The MCF-7 cells with stable expression of Cre recombinase were transfected withpStop-eGFP after selection of hygromycin B. There were green fluorescent protein exrpression of eGFP analyzed byfluorescence microscope after transfected by the t wo vectors . No eGFP positive cell was detected in MCF-7 cells andPEF transfected with pStop-eGFP only . Conclusion The recombinant vector Cre-pCEP4 can express Crerecombinase which can recognize loxP sites and delete the DNA frag ment bet ween t wo loxP sites in the sa meorientation .