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Their step-wise integration/selection ensures independence of the constitutive transactivator expression unit from the TRP driven regulated gene expression, thereby enabling the selection of highly regulated clones. Stable tet-controlled gene expression requires the transfer of both (r)TA and TRP into the target cell. The opposite is true for the reverse transactivators rtTA2s-M2 and rtTA-3 in the Tet-on system. In the Tet-off system, the tTA is released from its DNA binding site in the presence of doxycycline (Dox), a tetracycline derivative, thus abolishing gene expression. Two transactivator variants have been developed, differing primarily in their response to the effector molecule tetracycline. The transactivator binds with high affinity to the tetR-moiety of the TRP, a minimal promoter physically linked to the tet-operator sequence. It allows effector dose-dependent regulation and consists of two components, a tetracycline controlled transactivator (tTA) and a tet-responsive promoter (TRP) regulating the gene of interest. The most commonly applied gene regulation system is the tetracycline inducible gene expression (tet-) system, originally described by Gossen and Bujard. Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations.

Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.

Further differences were observed regarding induction kinetics and dose–response.

Resultsĭetermination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. The effect on tet-controlled gene expression was analyzed for retroviral “All-In-One” vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter. However, disadvantages of this strategy for integration into primary cells led us to develop an “All-In-One” vector system, enabling simultaneous integration of both components. Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations.

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