Maximum control over transgene expression is provided by inducible promoters that can be switched on and off according to need, usually by controlling the supply of a particular chemical ligand. Typically, the transcription factors that regulate such promoters are structurally modified by this ligand. Naturally inducible promoters have been used, but more robust inducible promoter systems have been developed either by using non mammalian, often bacterial, components, or by engineering mammalian proteins so that they are incapable of responding to endogenous inducers. Two widely used systems for regulating inducible transgene expression are described below.
Tetracycline-regulated expression
Here, expression is regulated at the transcriptional level. Two transgenes are involved. One has an E. coli tetR gene to constitutively express the Tet repressor protein. The other has the sequence of interest (usually a coding DNA) and an upstream promoter with the tetO operator in the intervening space. By specifically binding to the tetO sequence, the Tet repressor protein blocks expression of the downstream coding sequence. Gene expression can be restored, however, by providing doxycycline, a tetracycline analog that binds to the Tet repressor causing it to change its conformation and be removed from the tetO sequence (Figure 1A). Because expression is induced at the transcriptional level rather than at the protein level, a significant delay may occur before expression is induced in response to providing or removing the induction signal.
Fig1. Inducible expression of transgenes. (A) Tetracycline-inducible expression. A tetO operator inserted just upstream of transgene A is a recognition sequence that can be specifically recognized and bound by the E. coli Tet repressor protein (TetR). When constitutively expressed from a separately introduced tetR transgene, the Tet repressor protein binds to the tetO operator, and so prevents the expression of transgene A. However, if doxycycline (an analog of tetracycline) is subsequently provided, it binds to the Tet repressor protein, causing it to change its conformation so that it is no longer capable of binding the tetO operator. As a result, expression of the previously silenced transgene A is switched on. Note that toxicity effects reflecting high-level constitutive expression of the repressor limit the use of this system in some cells. (B) Tamoxifen-inducible expression. Here, coding sequences for the ligand-binding domain of a mutant mouse estrogen receptor (ER) are fused to a transgene of interest (TG) in an expression vector. The expressed ER–TG fusion protein is bound by the Hsp90 inhibitory protein complex, preventing it from performing its function. The mutant ER ligand-binding domain is not recognized by estrogen, but will bind to tamoxifen, an estrogen analog. Following tamoxifen binding, the fusion protein is released from the Hsp90 complex, and the protein of interest is activated, even though it is part of a fusion protein. P, promoter.
Tamoxifen-regulated expression
Where rapid induction and rapid decay of gene expression are essential, an inducible system that works at the protein level is needed. The estrogen receptor is normally bound by the Hsp90 protein inhibitory complex, keeping it inactive within the cytoplasm until its ligand, estrogen, enters the cell. Estrogen binds to the C-terminal ligand binding domain of the estrogen receptor, causing a change of conformation. As a result, the Hsp90 inhibitor is released and the activated estrogen receptor translocates to the nucleus to activate certain target genes.
Tamoxifen-inducible expression uses a mutant ligand-binding domain of the mouse estrogen receptor, Esr1. The mutant domain does not bind its natural ligand estrogen at physiological concentrations, but will bind the synthetic ligand 4-hydroxytamoxifen. A cDNA for the mutant Esr1 ligand-binding domain is fused to the coding sequence of a gene of interest. Hsp90 binding prevents expression of the fusion protein until tamoxifen is provided (Figure 1B).
