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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 a Cre-pCEP4 vector expressing Cre recombinase and verify that it can effectively identify loxP site, providing a basis for the establishment of an animal model of human disease. Methods: The recombinant vectors Cre-pCEP4 and pStop-eGFP were constructed. Fugene HD was transfected into porcine embryonic fibroblasts (PEF) and MCF-7 cell lines, and the expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. Results: The recombinant vectors Cre-pCEP4 and pStop-eGFP were successfully constructed and the two vectors were transiently co-transfected into PEF. The pStop-eGFP was transiently transfected into MCF-7 cells with cre recombinase and transiently transfected with pStop-eGFP Under the fluorescence microscope, the expression of green fluorescent protein was observed in both of the two kinds of cells. However, no expression of GFP was detected in MCF-7 cells and PEF transfected with pStop-eGFP alone. CONCLUSION: Recombinant vector Cre-pCEP4 can express Cre recombinase in cells and the expressed Cre recombinase can recognize loxP site and delete the DNA fragment between two loxPs.