tTA

Tetracycline-controlled transactivator (tTA) is a fusion protein used to regulate gene expression in a reversible, inducible manner. It combines the Tet repressor (TetR) DNA-binding domain with a transcription activation domain, typically VP16, enabling tTA to activate transcription from a tetracycline-responsive element (TRE or tetO) upstream of a minimal promoter.

In the Tet-Off configuration, tTA binds to tetO and drives expression of the target gene when doxycycline is absent. The addition of doxycycline binds tTA, causing a conformational change that prevents binding to tetO and shuts off transcription. This system allows temporal control of gene expression; turning doxycycline on or off can rapidly repress or resume transcription.

Components typically include: a tTA expression cassette under a chosen promoter (ubiquitous or tissue-specific), a responder construct with tetO-driven promoter controlling the gene of interest, and doxycycline as the inducer. Researchers often use enhanced promoter elements such as TRE-Tight to reduce background expression.

Variants and related systems include tTA2, which offers reduced basal activity and improved performance in mammalian cells, and the reverse transactivator rtTA (Tet-On), which activates transcription only in the presence of doxycycline. These tools are widely used in mammalian genetics, neuroscience, developmental biology, and cancer research for inducible gene expression in cell culture and transgenic models.

Advantages of the tTA system include rapid, reversible control and compatibility with various promoters. Limitations can include background leakiness, potential doxycycline effects in some contexts, and possible toxicity from high-level tTA or VP16 activity.