Transcriptional activator TGV mediates dexamethasone‐inducible and tetracycline‐inactivatable gene expression

Abstract

A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (P Top10 ) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven P Top10 reach from undetectable levels of the reporter enzyme b-glucuronidase in the absence of dexamethasone to induced levels reaching 15±20% of the Cauli¯ower Mosaic Virus 35S promoter (P CaMV35S ). By modifying the sequence of P Top10 , we generated a new target promoter (P Tax ) that is stably expressed over several generations and that can be activated to levels comparable to P CaMV35S , while yielding only slightly elevated background activities.

Regulation and Processing of Maize Histone Deacetylase Hda1 by Limited Proteolysis

“…To this end, transcription experiments were performed using the tetracycline (TET) system (Bohner et al, 1999) (Figure 8). …”

“…Because the reporter construct is integrated stably into the tobacco genome (tobacco TAX transgenic line), a physiological chromatin structure can be expected (Bohner et al, 1999). Cotransfection of plasmids expressing the activator protein TetR-VP16 (Triezenberg et al, 1988) and a putative TetR-fused repressor into tobacco protoplasts results in the competition of the two proteins for the TET sites and provides a measure for transcription repression activity.…”

“…Cotransfection of two plasmids with the 35S promoter driving the expression of the VP16 transcriptional activator and the protein of interest, respectively, both fused to the DNA binding domain of TetR, results in the competition of the two fusion proteins for the TET binding sites. The effect on transcription is monitored by determining GUS activity levels (Bohner et al, 1999). (B) ZmHda1 constructs.…”

“…Transcription experiments were performed using the tetracycline (TET) system, based on the targeting of proteins fused to the DNA binding domain of the TET repressor (TetR) to a synthetic promoter containing four TET operators upstream of a minimal promoter (Bohner et al, 1999). Four different constructs of ZmHda1 fused to TET-B/E were made to be tested for transcription repression activity in tobacco protoplasts: DAg (Taunton et al, 1996).…”

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

“…In order to achieve an optimal expression of transgenes with minimum undesirable effects, it is highly desirable to regulate the expression of transgenes in a controllable fashion. A number of chemical inducible gene regulation systems, or gene switches, have been developed based on a diverse collection of non-plant regulatory elements that respond to a variety of chemicals (Gatz et al 1992;Wilde et al 1992;Williams et al 1992;Mett et al 1993;Rieping et al 1994;Weinmann et al 1994;Aoyama and Chua 1997;Caddick et al 1998;Bohner et al 1999;Martinez et al 1999a, b;Bruce et al 2000;Padidam et al 2003). A chemical inducible gene regulation system that specifically regulates transgene expression in response to an exogenous inducer at a particular stage of plant development or in a specific organ is very valuable when using transgenes whose constitutive over expression is detrimental or lethal to the plant.…”