An Improved Tet-on System to Tightly Conditionally Regulate Reporter Gene Expression.

Yanping Ren, Xiangping Li, Qingyou Liu, Yanfei Deng, and Deshun Shi


Reporter genes are often used as markers totrack the integration and expression of target genes inanimal genetic engineering. To avoid potential side effectsfrom reporter genes, in this study an improved Tet-onsystem was developed to control reporter gene expression,and its effectiveness was explored in transgenic cells. First,the rtTA protein was fused with Tat and NLS proteins toobtain the prokaryotic expression vector pET32a-Tat-rtTA-NSL. A eukaryotic transgenic vector was constructed, p-HS4-BPA-TmA-HS4, in which the reporter (mCherry) andtarget (PRL) genes were promoted by TRE and BCN,respectively. After confirming the functionality of thetransgenic vector, purified rtTA protein and Dox wereadded to induce expression of the mCherry gene. Theoptimal amount of purified rtTA protein, its influence ontarget gene expression, and the time of rtTA protein actionwere each investigated separately. The results showed thatrtTA protein was expressed in transformed E. coli with IPTGinduction. TRE could promote mCherry gene expressionby co-transfecting the constructed transgenic vector andprtTA plasmid. When purified rtTA protein and Dox wereadded, red fluorescence was observed in Bcap-37 cellstransfected with the p-HS4-BPA-TmA-HS4 vector, and theexogenous PRL gene was expressed regardless of mCherrygene expression. The optimal amount of rtTA protein was16µg/mL, and it needed about 6 h to promote mCherrygene expression in transfected cells. These resultsdemonstrate that the expression of the mCherry reportergene can be tightly and conditionally regulated by our Tet-on system.

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