Tet B or not tet B: advances in tetracycline-inducible gene expression.

Be it the B class, or another class of tetracycline (tet) repressor, the utility and specificity of transcriptional regulators based on this family of prokaryotic DNA binding proteins is unparalleled. A method for regulating gene expression at will in mammalian cells has long been the holy grail. Transfections of uncontrolled numbers of plasmids and unregulated gene expression were breakthroughs in the early days of molecular biology when genes encoding abundant proteins first were introduced into cultured cells. Gone are those days and those antiquated and limited methods. Fine tuning is now essential. We need systems in which gene expression can be repressed and then induced at will. Such control is essential for products that are growth inhibitory or toxic, for example, components of the apoptotic cascade. We need to be able to monitor different levels of gene expression during discrete time periods in cultured cells and in animals to understand the regulation of signal transduction that culminates in different cell fates. Cells that stably express deleterious proteins or cytokines may be lost or their phenotype altered during long-term selection. Clearly, for gene therapy, regulation is crucial. Modulating gene expression in cycles that mimic endogenous patterns is highly desirable, and avoiding toxic levels is a must. Several regulatory systems exist, but the advantages of the tet system are remarkable and many. Because the critical regulatory elements are prokaryotic, this system has no effects on host genes. The absence of pleiotropic effects greatly simplifies the interpretation of the phenotype observed. Moreover, at the doses used, the tet system lacks toxicity in mammalian cells. Because tet is an antibiotic with a long history, extensive documentation exists as to its safety in humans, making it an excellent candidate for gene regulation in gene therapy. The independent and collaborative work of Bujard and Hillen and their colleagues (1, 2) first demonstrated the efficacy of this remarkable regulatable system in mammalian cells. In its initial and simplest rendition, removal of tet causes a bacterial tetracycline repressor (tetR) fused to a viral VP16 transactivator to induce gene expression via seven tandem copies of a tet-operator DNA (tetO) binding site juxtaposed to a minimal promoter. This interaction results in induction of gene expression of up to 5 orders of magnitude. Since then, refinements have greatly improved the versatility and applicability of this system, see for instance refs. 3–5. The tet inducer acts as an allosteric effector and becomes an integral part of the transcription factor, and there are no intermediate steps. As a result, the concentration of the inducer directly correlates with the concentration of the transcriptional activator, a feature that differs from all other inducible systems documented to date. As a result, the tet system could be used to test a long-held assumption that transcription is governed by an all-or-none mechanism once transcription factors reach a critical threshold (Fig. 1A). Support for this hypothesis derived from in vitro transcription studies (6, 7). These results also were supported by results obtained in intact live cells in which inducers such as cytokines activated maximal expression of reporter genes on reaching a critical concentration (8–10). These results were not unexpected as the notion that inducers only act once a threshold concentration has been reached was widely accepted and made eminently good sense. How else would the sharp boundaries of gene expression characteristic of early Drosophila development be achieved? How would cells determine when and whether to divide? The consequences of a mechanism that could give a partial effect, for example a range of transcription levels that could vary with the level of the inducer, might well be developmentally devastating. The simplicity of the tet system allowed a stringent test of the hypothesis that transcription could occur in a graded, rather than a threshold manner (3). The effects of different concentrations of inducer on the expression of a reporter gene were examined at the single cell level by using the fluorescence activated cell sorter. Two retroviruses encoding either the transactivator or a tet-inducible promoter driving expression of a message encoding green fluorescent protein (GFP) were introduced into a polyclonal population of thousands of cells. The results showed a graded response: GFP levels in individual cells containing a single copy of the reporter construct were proportional to the concentration of doxycycline (dox, a tet analog) in the medium. These results showed that a graded transcriptional response is possible (Fig. 1 A). However, in nature, as in most of the systems studied above, graded responses are likely to be rare. Instead, complex interactions involving more than one transcriptional regulator (H.M.B. and F.M.V.R., unpublished data) or a series of signal transduction steps from inducer to transcriptional activator, (see for example the mitogen-activated protein kinase cascade; ref. 11), which can serve to convert a given dose of inducer into a fine-tuned all-or-none response are likely to be the rule. Nonetheless, the finding that transcription can be induced to different levels is both of fundamental mechanistic interest and important experimentally, as it provides a means for controlling and studying the effects of precise doses of regulators on growth and differentiation in a manner previously not possible. A major advance in broadening the utility of the tet system was the use of retroviruses. Although proof of principle was established by using plasmids, this approach definitely involved a labor of love. First, the transactivator plasmid had to be transfected, and then stable clones were selected and tested for their responsiveness to tet by using a second plasmid containing an inducible marker gene. Once the few clones that adequately increased marker gene expression in response to tet were obtained, they were transfected with a third plasmid bearing the inducible gene of interest, and the process of clonal selection and testing was repeated. The yield after several months of cell culture often was a handful of well-regulated clones. If lucky, these did not drift over time, a feature that proved to be highly dependent on cell type, with HeLa cells providing perhaps the most stable tet responders (12). Because retroviral gene delivery is far more rapid and efficient than stable plasmid transfection and less variable than transient transfection, many investigators turned to tet-