荧光定位法快速准确筛选一种GFP重组质粒细胞株  

A Transgene Cell Line with GFP Recombinant Plasmid can be Screened Rapidly and Accurately by Fluorescent Localization Method

作　　者：邓玉风[1] 黄亿元 胡世凤[1] 黄媛恒[1] 路文盛[2] 黄勤[1] 

机构地区：[1]广西医科大学生理教研室,南宁530021 [2]广西壮族自治区人民医院内分泌科,南宁530021

出　　处：《基因组学与应用生物学》2016年第5期1060-1064,共5页Genomics and Applied Biology

基　　金：国家自然科学基金项目(No.81160102)资助

摘　　要：筛选脂质体介导的稳定转染细胞株的方法需要较多重复工作才能完成,尤其是转染效率不高的细胞。本研究报道一种快速准确获取稳定转染细胞株的方法,供同类实验参考。我们构建的真核表达载体带有可用于筛选阳性克隆的绿色荧光基因和Neo基因,采用脂质体将其转染入C2C12细胞,并用G418筛选阳性克隆,荧光显微镜下挑选绿色荧光与目的基因表达的蛋白在细胞内定位一致的细胞株,Western Blot可检测到融合蛋白表达,免疫共聚焦显示目的蛋白表达量增加;荧光散在分布于整个细胞的细胞株,经Western Blot检测没有融合蛋白表达。因此,根据GFP绿色荧光分布的位置可以准确挑选带有目的基因重组质粒的稳定细胞株。Searching a stable transfected cell line via liposomes needs much duplication of work, especially for cells with low transfection efficiency. This paper reports a rapid and accurate method to obtain stable transfection cell lines, providing reference for other similar investigations. We constructed the expression vectors carrying green fluorescent gene and Neo gene for screening positive clones, and subsequently transfected the vectors into C2C12 cells and screened positive clones in the presence of G418, then we selected clones containing green fluorescent under the fluorescence microscope. The results showed that in the selected cell lines whose location of green fluorescent was consistented with proteins expressed by target genes, the fusion protein expression was observed by Western blot, and confocal immunomiscrocopy indicated the expression of the target protein was increased. On the contrary, in the cell line whose fluorescence was distributed in the whole cell rather than just in the specific location of the cells, the fusion protein was not be observed by Western blot. Therefore the stable transgene cell lines may be screened rapidly and accurately according to the location of GFP in the cells.

关 键 词：稳定转染 细胞株 脂质体 荧光蛋白 

分 类 号：Q78[生物学—分子生物学]