LacZ基因在哺乳动物细胞中的稳定表达及其检测方法

Stable Transfection of LacZ Gene in Mammal Cells and Practical Detecting Assay for LacZ Gene Expression

目的 构建含有LacZ标记基因的真核表达载体和稳定表达LacZ的转基因细胞 ,并建立简易有效的检测方法。方法 采用脂质体转染法将装有LacZ基因的真核表达载体 pcDNA3导入鼠成纤维细胞L92 9,G418筛选LacZ基因稳定表达细胞株 ,并应用超声波破碎和贴壁细胞悬浮培养的方法来检测转基因细胞的转染率及目的蛋白的表达。结果 成功构建了稳定表达LacZ基因的转基因细胞 ,超声波破碎后其上清与 β-半乳糖苷酶 (LacZ)的底物X - gal反应显兰色 ;铺有固体琼脂粉的平皿中细胞培养至第 4天开始显兰色 ,并随时间的延长 ,比率增加 ,最终可高达 10 0 %。结论 LacZ基因在哺乳动物细胞中可稳定表达 。

Objective To construct a cell line stably expressing the LacZ gene, and to establish practical assay for detecting LacZ gene expression in mammal cells.Methods Mammal expression vector pcDNA3 carrying the LacZ gene was transfected into the mouse fibroblast cell line L929, and the cell clones expressing LacZ protein were selected. Stable transfected cells were verified by either disrupting the cell by sonification or culturing them on the solid agar with X-gal.Results The expected cell line was constructed successfully, LacZ protein in the cell lysate of the gene transfected cells was able to react with its substrate, (X-gal). and showed blue color .The cells cultured on the solid agar turned into blue at the 4th day, the ratio of the blue cells increased during the following days and then reached 100% at the 10th day.Conclusion LacZ gene can be transfected into mammal cell lines, and target protein can be stably expressed. Both methods for detecting the LacZ protein expression are practical and effective. Key words:LacZ;?mammal cell;?stable expression