An improved Tet-on system to tightly conditionally regulate reporter gene expression

Abstract

Reporter genes are often used as markers to track the integration and expression of target genes in animal genetic engineering. To avoid potential side effects from reporter genes, in this study an improved Tet-on system 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 to obtain the prokaryotic expression vector pET32a-Tat-rtTA-NSL. A eukaryotic transgenic vector was constructed, p-HS4-BPA-TmA-HS4, in which the reporter (mCherry) and target (PRL) genes were promoted by TRE and BCN, respectively. After confirming the functionality of the transgenic vector, purified rtTA protein and Dox were added to induce expression of the mCherry gene. The optimal amount of purified rtTA protein, its influence on target gene expression, and the time of rtTA protein action were each investigated separately. The results showed that rtTA protein was expressed in transformed E. coli with IPTG induction. TRE could promote mCherry gene expression by co-transfecting the constructed transgenic vector and prtTA plasmid. When purified rtTA protein and Dox were added, red fluorescence was observed in Bcap-37 cells transfected with the p-HS4-BPA-TmA-HS4 vector, and the exogenous PRL gene was expressed regardless of mCherry gene expression. The optimal amount of rtTA protein was 16 μg/mL, and it needed about 6 h to promote mCherry gene expression in transfected cells. These results demonstrate that the expression of the mCherry reporter gene can be tightly and conditionally regulated by our Tet-on system.