Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

TLDR

Control elements of the tetracycline‑resistance operon from E. coli Tn10 have been used to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor to the activating domain of HSV‑1 VP16, a constitutively expressed tetracycline‑controlled transactivator (tTA) was created that drives transcription from a minimal CMV promoter containing tet operator sequences.

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.