反义四环素转录激活子表达细胞系的构建及鉴定

Expression of rtTA in NIH 3T3 Cell Line

目的:构建反义四环素转录激活子(reverse Tc-controlled transactivator,rtTA)表达细胞系。方法:将RevTet-On基因表达系统中调控载体pRevTet-On转染NIH3T3细胞,经G418筛选及RT-PCR鉴定。结果:获得7个rtTA不同表达水平的细胞克隆。结论:成功构建rtTA表达细胞模型,为进一步研究特定外源基因的功能提供一个理想实验平台。

Objective To construct the Tet/3T3 cell lines that constitutively express the reverse Tc-controlled transactivator(rtTA).Methods Regulating plasmid pRevTet-on was transfected into NIH 3T3 cells and screened by G418 to construct the single-stable Tet/3T3 cell line,the positive clones which express rtTA were determined by RT-PCR.Results 7 cell clones expressing high level of rtTA were established.Conclusion The Tet/3T3 cell line is successfully reconstruction,which provides an ideal experimental platform for understanding the function and Tc-controlled expression of genes.