InvivoGen introduces a new and improved Tet-on system, specifically designed for the controlled expression of cytotoxic proteins.
Conditional expression of proteins in mammalian cells is favored over stable expression in a variety of basic and applied research areas, including functional genomics, gene therapy, biopharmaceutical protein production, and drug screening. Depending on experimental requirements, researchers can efficiently activate or suppress a gene of interest (GOI) at will, using different systems based on the E. coli Tn10-specified tetracycline-resistance operon [1].
InvivoGen offers such a system, named TiGer (Tetracycline-induced Gene expression or repression) system, which comprises two RepTor™ cell lines and two series of pTiGer plasmids (cloning and control vectors):
- RepTor™ cell lines: HEK293 and A549 cells constitutively expressing the tetracycline repressor protein (TetR)
- pTiGer-mcs plasmids: Cloning of a gene of interest (GOI) downstream of a Tet-inducible promoter
- pTiGer-reporter plasmids: Tet-inducible expression of a reporter gene (controls)
Features
The TiGer system has been specifically designed to allow the controlled expression of cytotoxic genes.
- Minimal GOI background expression without tetracycline
- Strong GOI expression with tetracycline
TiGer Tet-on assay principle
InvivoGen's TiGer Tet-on system relies on the combined use of two plasmids; the first expressing an optimized TetR construct, and the second, pTiGer, a GOI under the control of an engineered tetracycline operator (tetO)-containing promoter. It features the strong composite CMV-EF1 promoter modified to integrate a tetracycline response element (TRE).
The TetR plasmid was stably transfected into the RepTor™ cell lines, to save you time and effort. Upon transfection of the RepTor™ cells with a pTiGer-GOI plasmid, TetR binds to TRE and blocks the transcription of the GOI [2]. Addition of doxycycline, a synthetic analog of tetracycline, leads to the release of TetR from the tetO sequences, resulting in the derepression of the CMV-EF1-TRE promoter and transcription of the GOI.
The TiGer Tet-on system has been engineered to guarantee maximal repression of the GOI, ensuring minimal leakage of GOI expression in the absence of tetracycline/doxycycline, and strong GOI expression in the presence of the antibiotic. This key feature confers the TiGer Tet-on system with the advantage of working with cytotoxic genes (e.g. proteases, pore-forming proteins, gain-of-function variants).
Applications
InvivoGen’s TiGer Tet-on system is useful for, but not restricted to the following applications:
- Controlled expression of cytotoxic genes
- Fine-tuned expression of a GOI
- Screening of toxic protein inhibitors
References:
1. Kallunki, T. et al., (2019). How to choose the right inducible gene expression system for mammalian studies? Cells. DOI: 10.3390/cells8080796.
2. Hillen, W., Wissmann, A. (1989). Tet repressor-tet operator interaction. Protein-Nucleic Acid Interaction. DOI: 10.1007/978-1-349-09871-2_7.
