Lim K, Chae C B
University of North Carolina.
Biotechniques. 1989 Jun;7(6):576-9.
Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.
通过直接检测包含大肠杆菌β-半乳糖苷酶基因的重组基因所编码的β-半乳糖苷酶活性，能够轻松测定不同细胞类型的转染效率以及克隆基因的启动子强度。可以将β-半乳糖苷酶的底物邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）添加到含有转染细胞的培养皿中，然后测定从完整细胞或培养皿中裂解的细胞释放出的有色酶产物的强度。通过该检测获得的结果是对转染效率以及导入细胞中的基因的启动子强度的可靠衡量。此外，在用X-gal孵育后，可以在光学显微镜下鉴定并定量表达转染基因的细胞。因此，在评估DNA转染时，使用大肠杆菌β-半乳糖苷酶基因作为报告基因比使用氯霉素乙酰转移酶基因更方便。
