Tight control of gene expression in mammalian cells by tetracycline-responsive promoters
Existing inducible mammalian promoters were leaky or used slow inducers. This study fused the E. coli tet repressor to VP16, creating a transactivator that drives tet-operator promoters, silenced by tetracycline. Withdrawal activated a linked gene by five orders of magnitude.
Parts used
Key findings
- Established the foundational Tet-Off inducible expression system in mammalian cells. The tet repressor from E. coli Tn10 was fused to the VP16 transactivation domain to create the tetracycline-controlled transactivator (tTA). tTA drives transcription from a minimal CMV promoter combined with tet operator (tetO) sequences. In tTA-expressing HeLa cells, the luciferase reporter was induced >10,000-fold above background. Critically, adding tetracycline (or doxycycline) suppresses tTA binding and shuts off expression within hours, giving tight, reversible transcriptional control. This tTA/TRE (tetracycline response element) architecture became the canonical Tet-Off vector design used across virtually all inducible transgene systems.
Abstract
Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia 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 microgram/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.