Fig. 1.
Schematic representations of the five different Tet-regulatable systems. (A) In general, Tet-regulatable systems are composed of two distinct expression cassettes, one containing the regulatory protein (tTA, rtTA, tTS) under the control of a ubiquitous cell type specific promoter (cassette A) and the second cassette containing the Tetracycline Responsive Element (TRE) fused to a promoter, regulating the expression of the reporter or therapeutic transgene (cassette B). (B) The chimeric protein tTA is composed of the TetR fused to the viral transactivator VP16. In the absence of Dox, the tTA protein binds to the TRE within the compound promoter PCMVmin to allow the expression of the transgene. In the presence of Dox, the tTA changes its conformation, gets detached from the TRE and no transgene expression is observed. (C) The rtTA transactivator is also a chimeric protein composed of the TetR and the VP16 domains. However, due to the presence of four mutations in the TetR (now called rTetR) domain, this protein fixes the TRE in the presence of Dox, thus allowing transgene expression. In the absence of Dox, rtTA gets detached from the TRE and no transgene expression is observed. (D) The chimeric protein tTS is composed of the TetR fused to the KRAB box of Zink finger proteins. In the absence of Dox, tTS binds to the TRE and inhibits transgene expression driven by a normal promoter (i.e. CMV). In the presence of Dox, tTS gets detached from the TRE, thus allowing transgene expression. (E) The combined TetOn system contains both the rtTA and the tTS protein, simultaneously expressed from cassette A. In the absence of Dox, the tTS protein binds to the minimal compound promoter and inhibits even the low background expression, while the rtTA protein stays detached from the TRE. In the presence of Dox, rtTA binds to the TRE and activates expression of the transgene, while tTS stays detached. (F) In the autoregulatory loop system, both rtTA (or tTA) and the transgene of interest are driven by the compound minimal promoter separated by an internal ribosomal entry site (IRES). In the absence of Dox, no transgene is expressed, while in the presence of Dox, both transgenes, transactivator and gene of interest, are expressed. Onset of expression takes longer, because initially, sufficient amounts of rtTA need to be produced to completely activate the system.