Components of the Tet Switches (Gossen and Bujard, 1992) are derived from the tetracycline resistance operon in E.coli, one of the most evolved gene regulation systems. Antibiotics like tetracycline act by inhibiting bacterial translation and in order to survive, bacteria have developed mechanisms of resistance that prevent lethal intracellular antibiotic concentrations. Thus, Tet resistance determinants were optimized during evolution to interact with exquisite sensitivity and extraordinary specificity (Hillen and Berens, 1994). TetR, the Tet repressor protein, inhibits transcription of the resistance protein in the absence of the antibiotic by binding to tetO sequences in the promoter region of the resistance gene. The TetR protein senses sub-inhibitory tetracycline concentrations which is reflected by its extraordinary high binding constant for Doxycycline (Ka=1010 M-1). Binding of the antibiotic to TetR diminishes the affinity of the repressor protein to tetO sequences by 9 orders of magnitude resulting in repression relieve and expression of the resistance protein.
Because of this extremely high specificity of the TetR protein for its tetO DNA binding sites, TetR-based DNA binding proteins can specifically recognize their cognate binding sequences even in much more complex eukaryotic genomes which contain a much higher degree of competing unspecific DNA sequences.
While TetR acts as a repressor in bacteria, it is assumed that in eukaryotic cells transcriptional activators will confer tighter control of gene expression (less basal activity). Therefore, a strong eukaryotic transcriptional activator was generated by fusing the activating domain of the Herpes simplex virus VP16 trans-activator protein to tetR. To facilitate specific binding, its cognate binding site tetO was multimerized and embedded into eukaryotic minimal promoters thus creating the Tet-inducible gene expression system.
In 1992, the original Tet Expression System was first described. This Technology, now known as the Tet-Off system, supports expression in the absence of tetracyclines. Addition of tetracyclines prevents DNA binding and transcription of any gene of interest cloned behind the Tet-regulated promoter stops. Over the years both original components -transactivator and minimal promoter- have been modified and improved (Gossen et al., 1995; Urlinger et al., 2000; Zhou et al., 2006; Loew et al., 2010). A major breakthrough was the development of a complementary system based on a reverse transactivator (Tet-On) that requires binding of tetracyclines (e.g. Doxycycline) for promoting expression of genes controlled by Tet-responsive promoters. This original Tet-On transactivator was further improved to reduce residual binding to a responsive promoter in the absence of the effector and to increase its sensitivity to Dox (Urlinger et al., 2000).
In a most recent effort to improve the technology, the Tet-responsive promoter was modified to reduce basal expression in the absence of transactivators while maintaining high levels of induced expression (Loew et al., 2010). Combined with the latest Tet-On transactivator which is ten-fold more sensitive to the effector than previous versions (Zhou et al., 2006) this new Tet System was recently launched by Clontech as the ‘Tet-On 3G’ system.