A tetracycline-controlled transactivator (tTA) was generated by fusing the tetracycline repressor (tetR) with the activating domain of herpes simplex virus virion protein 16 (VP16). This tTA is constitutively expressed in HeLa cells and activates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator (tetO) sequences. When integrated into a tTA-producing HeLa cell line, luciferase expression is highly regulated by tetracycline, with activity varying over five orders of magnitude depending on tetracycline concentration. This system allows precise control of gene expression in mammalian cells and enables reversible "on/off" regulation of gene activity. The system is based on regulatory elements of the Tn10-specified tetracycline resistance operon of E. coli, where transcription is negatively regulated by tetR. In the presence of tetracycline, tetR does not bind to its operators, allowing transcription. By combining tetR with the C-terminal domain of VP16, a hybrid transactivator was generated that stimulates minimal promoters fused to tetO sequences. These promoters are virtually silent in the presence of low concentrations of tetracycline, which prevents tTA from binding to tetO sequences. The specificity of tetR for its operator sequence, the high affinity of tetracycline for tetR, and the well-studied properties of tetracyclines make this system superior to the lacR/O/IPTG system. This has been demonstrated in plant cells, where direct repressor action at promoter sites is efficiently reversed by the antibiotic. The tTA-dependent promoters were constructed by inserting tetO sequences upstream of minimal promoter sequences. These promoters were tested for their ability to be activated in transient expression experiments using the HeLa cell line HtTA-1. The heptameric version of the tetO sequences caused the highest activation of all Ptk-tetO constructs. The resulting plasmids pUHC13-3 and pUHC13-4 contain the heptameric tetOs in two orientations differing in the distance between the operators and position +1 of P_hCMV by 19 bp. The tTA system allows for precise control of gene expression in mammalian cells, with tetracycline concentrations varying from 0 to 1 μg/ml affecting luciferase activity over five orders of magnitude. The system is efficient, rapid, and reversible, making it suitable for studying gene function and mRNA decay rates under physiological conditions. The tTA system is also advantageous compared to the lac system, as tetR binds tetracycline much tighter than lacR binds IPTG. This allows for the use of very low, nontoxic concentrations of tetracycline. Additionally, the system is suitable for application in transgenic animals due to the availabilityA tetracycline-controlled transactivator (tTA) was generated by fusing the tetracycline repressor (tetR) with the activating domain of herpes simplex virus virion protein 16 (VP16). This tTA is constitutively expressed in HeLa cells and activates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator (tetO) sequences. When integrated into a tTA-producing HeLa cell line, luciferase expression is highly regulated by tetracycline, with activity varying over five orders of magnitude depending on tetracycline concentration. This system allows precise control of gene expression in mammalian cells and enables reversible "on/off" regulation of gene activity. The system is based on regulatory elements of the Tn10-specified tetracycline resistance operon of E. coli, where transcription is negatively regulated by tetR. In the presence of tetracycline, tetR does not bind to its operators, allowing transcription. By combining tetR with the C-terminal domain of VP16, a hybrid transactivator was generated that stimulates minimal promoters fused to tetO sequences. These promoters are virtually silent in the presence of low concentrations of tetracycline, which prevents tTA from binding to tetO sequences. The specificity of tetR for its operator sequence, the high affinity of tetracycline for tetR, and the well-studied properties of tetracyclines make this system superior to the lacR/O/IPTG system. This has been demonstrated in plant cells, where direct repressor action at promoter sites is efficiently reversed by the antibiotic. The tTA-dependent promoters were constructed by inserting tetO sequences upstream of minimal promoter sequences. These promoters were tested for their ability to be activated in transient expression experiments using the HeLa cell line HtTA-1. The heptameric version of the tetO sequences caused the highest activation of all Ptk-tetO constructs. The resulting plasmids pUHC13-3 and pUHC13-4 contain the heptameric tetOs in two orientations differing in the distance between the operators and position +1 of P_hCMV by 19 bp. The tTA system allows for precise control of gene expression in mammalian cells, with tetracycline concentrations varying from 0 to 1 μg/ml affecting luciferase activity over five orders of magnitude. The system is efficient, rapid, and reversible, making it suitable for studying gene function and mRNA decay rates under physiological conditions. The tTA system is also advantageous compared to the lac system, as tetR binds tetracycline much tighter than lacR binds IPTG. This allows for the use of very low, nontoxic concentrations of tetracycline. Additionally, the system is suitable for application in transgenic animals due to the availability