Diverse strategies for tetracycline-regulated inducible gene expression.

The recent development of tetracycline (tet)-regulated transactivation systems for inducible gene expression has dramatically enhanced the tools available for the temporal and quantitative control of exogenous genes in mammalian cells and transgenic mice and plants. Such systems have applications in many areas of biology and medicine, including the study of gene regulation and function in developmental systems; the role and biochemistry of particular genes in various biological processes; and the safe, controlled, administration of gene therapy. These systems have two central components: transcriptional transactivators that interact specifically with bacterial cis regulatory elements and antibiotics that modulate the binding of the transactivators at low, nontoxic doses. The consequence is a substantial reduction of nonspecific pleiotropic effects observed with earlier systems. Here we summarize the current status of the use of tet-regulated transactivation systems for the control of gene expression, including the contribution by Hoffman et al. in this issue of the Proceedings (ref. 1). The first tet-regulated gene expression system for use in mammalian cells, developed by Gossen and Bujard (2), involved constitutive expression of the tet transactivator protein (tTA) with the human cytomegalovirus (CMV) immediate early (IE) promoter/enhancer. tTA is a fusion protein composed of the tet repressor of Escherichia coli and the transcriptional activation domain of the VP16 protein of herpes simplex virus. In the absence of tet, the tet repressor portion of tTA mediates high affinity, specific binding to sequences from the tet resistance operator of TnlO (tetO). In the presence of tet, however, a conformational change in tet repressor prevents tTA from binding to its operator (3). Genes to be regulated by tTA (e.g., luciferase) were placed under the control of a hybrid, inducible promoter (hereafter referred to as tetP) which consists of a human CMV IE minimal promoter preceded by seven copies of tetO. In this initial study, performed in HeLa cells stably expressing tTA, expression of luciferase was very low in the presence of ng/ml quantities of tet, and removal of tet resulted in as much as a 100,000-fold increase in luciferase levels. Luciferase levels could be varied by titrating the amount of tet in the growth media, and maximal, steady-state levels of activity were achieved in about 24 h. Somewhat surprisingly, tTA was undetectable in the HeLa cells by Western blotting (although it was detected with a sensitive gel mobility shift assay), an observation consistent with toxicity of the tTA protein. This was speculated to be a consequence of transcriptional squelchirng, in which tTA would act as a sink for the general transcriptional machinery of the cell, resulting in the death of cells expressing moderate to high levels of tTA. Several technical and practical reviews of this system and its advantages over other inducible expression systems have appeared recently (4-6). This basic system, since its description, has been used extensively in tissue culture for the expression of a variety of different genes. HeLa cells stably expressing tTA have been used to study the consequences of tet-regulated, tetP-