Construction And Detection Of Bicistronic Tet-on Vectors

Tet-on system is an inducible gene expression system based on tetracycline operaon.When doxycycline(Dox),tetracycline derivatives,is absentthe reverse tetracycline-controlled transactivator(rtTA) is not able to recognize tetracycline operator(TetO), the target gene cann't be expressed;only when there is Dox,rtTA is able to recognize and combine with TetO,then promote the transcription of target gene.This research is mainly about the transformation of recent Tet-on system,making use of 2A fragment from FMDV and IRES from EMCV.Two single-plasmid,single-promoter and dual-reporter Tet-on vector were constructed, dsredl and egfp were used as reporter for quantitative detection. According to Real-time PCR and microscopy results, when Dox is absent,the background expression level of both two modified Tet-on systems is lower than that in the original system pTRE AutoR3,the induction multiple is also better. Among the two modified systems, the background level of pTRE DsRedl-IRES-EGFP AutoR3 is lower and the induction multiple is higher.These two expression systems not only can control the expression of target gene via Dox,but also detect its expression level via the fluorescence intensity of EGFP.If the upstream gene dsredl is replaced by functional gene,these modified systems will be able to control gene expression time and amount by Dox and its amount,also can detect gene expression by EGFP.It would be a supplement and innovation for Tet-on research,and make the operation of Tet-on more convenient.