【摘要】 目的探讨磷酸钙共沉淀法与阳离子脂质体试剂形成复合物法两种基因转染方法构建大鼠肝癌细胞克隆株的有效性及对生长影响的比较。方法将携带增强型绿色荧光蛋白的真核表达载体pEGFP-N1分别用脂质体形成复合物法、磷酸钙共沉淀法导入大鼠肝癌CBRH-7919细胞,G418筛选基因稳定转染阳性单细胞克隆株。RT-PCR法检测EGFP基因在细胞中的表达。结果两种方法转染CBRH-7919细胞24h后,倒置相差荧光显微镜下观察均见绿色荧光。G418加压筛选14d后均形成阳性克隆,倒置相差荧光显微镜下可见绿色荧光,脂质体法的细胞克隆荧光强度强于磷酸钙。阳性克隆株经RT-PCR法检测,750bp于450bp处均有特异性条带。结论两种细胞转染方法均可使增强型绿色荧光蛋白基因稳定转染CBRH-7919,为今后探讨外源目的基因对靶细胞生长特性的影响奠定前期实验基础。更多还原

【Abstract】 Objective To study the effect of gene transfection with cationic liposome and calcium phosphate coprecipitate.Methods The foreign gene was transferred into rat hepatoma carcinoma cell with cationic liposome and calcium phosphate coprecipitate,whose influence on cell growth was observed.Cationic liposome transfection and calcium phosphate coprecipitate transfection were utilized to introduce the eukaryotic expression vector pEGFP-N1 containing enhanced green fluorescent protein gene in rat hepatoma carcinoma cell CBRH-7919,respectively.Screen positive cell clones were selected by G418.GFP gene expression was detected with RT-PCR.Results CBRH-7919 were transfected with the two transfection methods.The green fluorescence could be observed under inverted optical microscope.The positive stable cell clones were obtained by G418 growth inhibition screen 14d after transtection.Conclusion Both cationic liposome and calcium phosphate coprecipitate can stablely transfect enhanced green fluorescent protein gene in CBRH-1719.更多还原