Improved single-chain transactivators of the Tet-On gene expression system

We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.Inducible gene regulation systems that utilize small, non-toxic effector molecules to control transgene expression in eukaryotic cells and organisms have become invaluable tools in many biological research areas, such as functional genomics and gene therapy. Among the currently used regulatory circuits, the Tet-Off and Tet-On systems based on the tetracycline-resistance determinant from transposon Tn10 are the most widely applied and best studied [1-3]. In Escherichia coli, the Tet repressor protein (TetR) forms a dimer that binds to the tet operator (tetO) DNA sequence with high affinity and specificity. Tetracycline (Tc) or its derivative doxycycline (dox) binds to TetR and triggers a conformational change that prevents the protein from binding to tetO. Fusion of TetR to the VP16 activation domain of herpes simplex virus resulted in the Tc-responsive transactivator (tTA) which has retained the DNA-binding specificity and effector-inducibility of TetR [4]. In the absence of effector, tTA binds to tetO and activates transcription from an appropriately-positioned minimal promoter. Administration of Tc or dox switches gene exp