The tetracycline-responsive promoter contains functional interferon-inducible response elements

Abstract

Tetracycline (tet)-responsive expression vectors allow controlled inducible expression of proteins in mammalian cells. This system is widely used for experimental research both in vivo and in vitro. In our attempts to use this system to study the antiviral effect of IFNalpha on hepatitis B virus, we discovered an unexpected feature of the tet-responsive promoter (tet promoter) of the currently available expression vectors. IFNalphawas found to stimulate tet promoter activity after transient transfection in a dose- and cell type-dependent manner. By sequence inspection, an IFNalpha-stimulated response element (ISRE)-like sequence was identified in the linker regions located between the heptameric tet operator sequences. Gel shift assays revealed binding of IFN-stimulated gene factors to these sequences, indicating that they mediate the IFNalpha-mediated promoter stimulation. These data demonstrate an unexpected feature of the tet-responsive expression system which needs to be taken into account when using this system for analysis of cytokine functions in vitro and in vivo. The data also imply that the tet promoter-based expression system can be rendered non-responsive to IFNalpha by mutagenesis of the ISREs and this may be essential when considering gene therapy in vivo.

Liver-specific expression of interferon γ following adenoviral gene transfer controls hepatitis B virus replication in mice

“…42 In comparison to vectors containing GFP, mIFNb, or mIFNg transgenes, we observed a two-to five-fold increased basal level of transgene expression in mice infected with Adbiluc-mIFNa (Figure 4). This may be due to IFN responsive elements located in the linker regions between the heptametrical Tet operator sequences in P tet , 43 which in novel versions of this promoter have been eliminated (H Bujard, unpublished results).…”

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

“…Second, which minimal promoter is most appropriate for transgene expression? The original minimal promoter P hCMV*-1 24 displays some cell-type specific leakiness 30 , due in part to the presence of functional interferon-α inducible response elements 29 . This led to the development of second generation Tet-dependent promoters, termed ΔMtetO, TREtight and SG-TRE, with altered spacing and sequences between the tetO elements and with different minimal promoter sequences 33,[40][41][42] .…”

“…It only binds to the TRE and, concomitantly, activates transcription in the presence of doxycycline. Residual leakiness of the system, i.e., transgene expression in the absence of TRE-bound transcription factor, originating either (i) from position effects at a genomic integration site, (ii) from the TRE itself 29 , or (iii) from non-specific binding of tTA/rtTA 28 , was addressed by introducing an additional transcriptional silencer, termed tTS (tetracycline-dependent transcriptional silencer) 30 to the system. It forms a dual regulator network together with rtTA (see Figure 1).…”

Anticancer activity of oncolytic adenovirus vector armed with IFN-α and ADP is enhanced by pharmacologically controlled expression of TRAIL

“…In DLD-1 cells, the KD3-IFN helper provided slightly but significantly higher SEAP expression than did KD3 (Po0.001). An IFN-a-stimulated response element-like sequence within the TRE of the TetON system has been reported, 36 suggesting that the expression of IFN-a by KD3-IFN could mediate various levels of SEAP expression in different cell lines.…”