Control elements of the tetracyclineresistance operon encoded in TnlO ofEscherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 pzg/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way. Here we describe a control system that in HeLa cells allows regulation of expression of an individual gene over up to five orders of magnitude. This system is based on regulatory elements of the TnlO-specified tetracycline-resistance operon of E. coli (19), in which transcription of resistancemediating genes is negatively regulated by the tetracycline repressor (tetR). In the presence of the antibiotic tetracycline tetR does not bind to its operators located within the promoter region of the operon and allows transcription. By combining tetR with the C-terminal domain of VP16 from HSV, known to be essential for the transcription of the immediate early viral genes (20), a hybrid transactivator was generated that stimulates minimal promoters fused to tetracycline operator (tetO) sequences. These promoters are virtually silent in the presence of low concentrations of tetracycline, which prevents the tetracycline-controlled transactivator (tTA) from binding to tetO sequences.The specificity of the tetR for its operator sequence (19) as well as the high affinity of tetracycline for tetR (21) and the well-studied chemical and physiological properties of tetracyclines constitute a basis for an inducible expression system in mammalian cells far superior to the 1acR/O/IPTG system. This has already been demonstrated in plant cells, in which direct repressor action at promoter sites is efficiently reversed by the antibiotic (22, 23). MATERIALS AND METHODSConstruction of the Transactivators tTA and tTAS. The tetR sequence was originally recovered from pWH510 (24) by PCR and inserted into pUHD10-1 (14), resulting in pUHD14-1 (A. Bonin and H.B., unpublished). A unique Aft II cleavage site overlapping the tetR stop codon in this plasmid construct allows for the in-frame insertion of coding sequences. To generate tTA, a 397-base-pair (bp) Mlu IlFok I fragment of pMSVP16 (20) coding for the C-terminal 130 ...

A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P tet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. T he repressor of the Tn10 tetracycline (Tc) resistance operon of Escherichia coli (TetR) recognizes its genuine operator (tetO) with unusual specificity (1). The interaction between repressor and operator is efficiently prevented by Tc, particularly by doxycycline (Dox) that binds to TetR with high affinity (2). These parameters, as well as the fact that Dox, a nontoxic compound widely used in medicine, readily traverses cell membranes, have made the elements of the tet resistance operon attractive for the development of a transcription control system that would function in higher eukaryotic cells. It was expected that, because of their evolutionary distance, the prokaryotic regulatory elements would not interfere with the metabolism of, e.g., a mammalian cell. Accordingly, it appeared feasible to superimpose onto the complex regulatory network of a cell an independent control circuit that could be governed from outside at will. Indeed, by fusing TetR with transcription activation domains, Tc controlled transactivators (tTAs) were obtained that efficiently activate P tet , minimal promoters fused downstream of an array of tetO sequences (3, 4). The presence of Dox would prevent this activation.A TetR mutant containing four amino acid exchanges of which three are located in the protein core, where inducer is bound and triggers the conformational change necessary for induction and where dimerization takes place (5, 6), exhibits a reverse phenotype when fused to a transcription activator (C-terminal portion of VP16 of herpes simplex virus) and examined in mammalian cells (7). This mutant, called rtTA, requires Dox or anhydrotetracycline for activation of P tet . Both Dox-controlled transcription activation systems, which operate in a complementary way, have been widely used in studies of gene function in various cellular systems, as well as in whole organisms including yeast, plants, Drosophila, mice, and rats (for review see ref. 8).Despite numerous successful applications, the currently available Tet regulatory systems show some limitations. Here, we focus on the previously described rtTA, which requires Dox or anhydr...

The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs.