Leadership In Controlled Gene Expression

The Tet Technology comprises two complementary control circuits, initially described as the tTA dependent [1] and rtTA dependent [2] expression systems. They are now commonly referred to as the Tet-Off System (i. e. tTA dependent) and the Tet-On System (i. e. rtTA dependent). In each system, a recombinant tetracycline controlled transcription factor (tTA or rtTA) interacts with a tTA/rtTA responsive promoter, P_tet, to drive expression of the gene under study (see Fig. 1 below). Expression is regulated by the effector substance tetracycline or one of its derivatives. Tetracyclines act at the level of DNA binding of tTA and rtTA transcription factors. rtTA requires a tetracycline ligand for DNA binding and hence, transcription. In sharp contrast, the interaction between tTA and DNA is prevented by tetracycline. Thus, the two versions of the Tet System respond to tetracyclines very differently, making them complementary as each system has its unique characteristics and strengths. High induction levels combined with low basal background expression result in an excellent dynamic range, a hallmark of both expression systems, allowing regulation of gene expression over several orders of magnitude without interference with the host cell physiology.

The Tet System owes these characteristics to:

• the prokaryotic origin of its core components, the Tet repressor, TetR, and its cognate binding site, the tet operator, tetO, making the system essentially inert in an eukaryotic environment
• the pharmacological properties of its effectors, in particular doxycycline (Dox), an intensely characterized antibiotic broadly applied in man and animals
• the favourable thermodynamic parameters of both the repressor/operator and repressor/effector interactions

As depicted in Figure 1, the two Tet dependent control circuits consist of four basic elements:

• tTA is a hybrid transcription factor resulting from the fusion of the prokaryotic Tet repressor, TetR, with a eukaryotic transcriptional transactivation domain (most widely used so far is the acidic domain of HSV VP16). The TetR moiety confers sequence specific DNA binding, sensitivity to tetracyclines and dimerization to the tTA fusion protein. Accordingly, the response of both TetR and tTA to tetracyclines is similar: binding of the antibiotic dramatically lowers their affinity to their common cognate binding sites, the tet operators.
• rtTA differs from tTA by a few point mutations within TetR. These, however, result in a complete reversal of tetracycline responsiveness of this transcription factor. rtTA requires tetracyclines for binding to tetO. Of note, specific tetracycline derivatives like doxycycline (Dox) or anhydrotetracycline (ATc) must be used to optimally exploit the rtTA phenotype.
• P_tet is a synthetic promoter responsive to both tTA and rtTA. It is comprised of a minimal RNA polymerase II promoter (transcriptionally silent in the absence of additional transcription factor binding sites) fused to multimerized tetO sequences (Figure 2). This arrangement makes the activity of P_tet dependent on the binding of tTA or rtTA. The design of such synthetic tTA/rtTA responsive promoters is flexible with respect to both the origin of the minimal promoter as well as the exact arrangement of the operators. The original version which consists of a CMV minimal promoter fused to an array of seven tetO sequences is designated P_tet-1. It is commercially distributed as part of the pTRE vector series (for tetracycline responsive element), somewhat in line with the prevailing eukaryotic nomenclature.
• Doxycycline, a tetracycline derivative, is currently the most preferable effector substance for both the Tet-On and the Tet-Off System.
  It binds with high affinity to tTA as well as to rtTA and, thus, is fully effective in the Tet-Off system at concentrations as low as 1-2 ng/ml in the case of tTA. In the Tet-On systems, concentrations as low as 80 ng/ml, in the case of rtTA2-syn1, are effective. An excellent medical safety record and well characterized pharmacological properties like excellent tissue penetration and low toxicity in eukaryotes make Dox the effector substance of choice for most applications in tissue culture and whole organisms.

Reiterated improvements of the components constituting the regulatory circuits resulted in expression systems that can be readily established (following a standard procedure), or conveniently adapted to special requirements thanks to the modular nature and compatibility of its elements.