Figure 1.

Illustration of the natural bacterial tetracycline resistance mechanism and synthetic tetracycline-based gene expression systems. A, In the absence of tetracycline (tet), the tet repressor (TetR) is bound to its cognate tet operator (tetO) DNA-binding motif, repressing the expression of the tet resistance-mediating tetA gene. Upon increasing cellular levels of tet, tet binding induces a conformational change of TetR, leading to its dissociation from the operator sequence, and expression of tetA ensues. B, The tet-OFF system designed for use in mammalian cells is based on a synthetic switch comprising the natural TetR fused to the activating domain of VP16 of the herpes simplex virus and a synthetic promoter with a series of repeats of the tetO motif placed upstream of a minimal promoter (e.g. human cytomegalovirus minimal promoter). The system is constitutively active and is turned OFF in the presence of the antibiotic. Implementation of a reversed TetR mutant (rTetR) generates a tet-ON system: tet induces the binding of rTetR to the target sequence, which in turn induces gene expression (tet can be replaced by other antibiotics of the tetracycline family like doxycycline). Replacement of VP16 by a transrepressor such as KRAB inverts the effect of the switch (not depicted here). GOI, Gene of interest. (Adapted from Gossen et al., 1995.)