高表达人cycli nG2基因的SGC-7901细胞克隆筛选和鉴定

Establishment and appraisement of human cyclin G2 gene over-expression human gastric carcinoma cell clones by lipofectin gene transfection

应用脂质体基因转染技术建立稳定表达cyclin G2基因的胃癌克隆细胞,为深入研究cyclin G2在胃癌细胞周期调控的作用和机制提供理想的生物学模型。构建以新霉素基因作为筛选标志基因的包含人cyclin G2基因真核重组表达载体pIRES-G2,大量扩增纯化后经限制性内切酶BamH I/BstX I双酶切鉴定;利用阳离子脂质体介导的基因转染法将其和对照空载体pIRESneo转染人胃癌细胞SGC-7901,经G418选择性培养基筛选后有限稀释法连续克隆化,免疫细胞化学染色检测cyclin G2蛋白表达情况。酶切结果表明cyclin G2cDNA已成功插入pIRESneo的多克隆位点内;经G418筛选后在转染pIRES-G2和转染pIRESneo组均见多个细胞克隆出现,细胞化学染色证实pIRES-G2转染组cyclin G2蛋白的表达水平明显高于pIRESneo转染组;经过多次有限稀释获得稳定高表达cyclin G2的细胞克隆。应用脂质体转基因技术成功建立高表达cyclin G2基因的人胃癌细胞克隆。

In order to build up a gastric carcinoma cell expressing human cyclin G2 gene stably, a useful tool for the research on the function of cyclin G2 in gastric carcinoma cell cycle regulation was made. The eukaryotic expression plasmid plRES - G2 containing cyclin G2 gene was constructed and identified by agarose gel electrophoresis after being cut with restricted endonuclease. The plRES - G2 and the empty vector plRESneo were introduced into human gastric carcinoma cell line SGC - 7901 using cation lipofectamine. Cell clones were obtained by G418 selective medium culture and limited dilution. The expression of cyclin G2 protein was detected by cytochemistry staining. BamH I / BstX I double restriction enzyme digestion expressed that a recombinant plasmid had been constructed successfully. Two cell clones expressing cyclin G2 stably were acquired after G418 selection and limited dilution. Cytochemistry staining results showed that the expression level of cyclin G2 in plRES - G2 transfection group was higher than that of plRESneo group. The result showed that cyclin G2 gene was stably and effectively expressed in obtained SGC- 7901 cell clones.