脂质体介导hHCN_2基因转染HEK293细胞的效率研究

Study of transfection efficiency of hHCN_2 into HEK293 mediated by LipofectamineTM 2000

目的:脂质体介导重组起搏基因pIRES2-EGFP-HCN2转染HEK293细胞,观察转染效率,探讨其构建生物起搏器的可行性。方法:对含hHCN2cDNA的PTR载体进行转化和扩增,将所得hHCN2基因定向克隆到真核表达载体pIRES2-EGFP中,双酶切来鉴定克隆的正确性。将重组质粒用脂质体转染HEK293,荧光显微镜观察EGFP的表达并计算转染效率。结果:构建了重组质粒pIRES2-EGFP-HCN2。荧光显微镜下可见转染后的HEK293呈绿色荧光,其转染效率可达90%以上。结论:成功构建了重组质粒pIRES2-EGFP-HCN2,并用脂质体转染的方法导入HEK293细胞,为生物起搏的研究奠定实验基础。

Objective： To investigate the feasibility and efficiency of human embryonic kidney ceils （HEK293） transfected with plRES2-EGFP-HCN2 by LipofectamineTM 2000. Methods：The PTR carrier with hHCN2 eDNA was converted and amplified, hHCN2 gene was cloned into the eukaryotic expression vector plRES2-EGFP,and the clone was identified by double enzymatic excision. The recombinant plasmid was transfeeted into HEK293 by LipofectamineTM 2000. EGFP expression was observed by fluorescence microscopy and the efficiency of transfection was calculated. Results：The recombinant plasmid was constructed. Transfected cells showed green fluorescence under a fluorescence microscope; the transfection efficiency was up to 90%. Conclusion ：Recombinant plasmid plRES2-EGFP-HCN2 is successfully constructed and transferred into HEK293 cell mediated by LipofectamineTM 2000,which will enable us to explore the role of HCNz gene in biological pacemaker.