Abstract
On account of its stringent regulation and high rate of induction, the tetracycline regulatory system is used extensively for inducing target gene expression in eukaryotes. However, under certain circumstances, its associated background expression can be problematic, as in the expression of highly toxic proteins. We found that when using the Tet-On 3G system to drive expression of the kid toxin gene in sf9 insect cells, a higher percentage of cells were killed than when using an empty vector in the absence of the induction agent doxycycline, thereby indicating the leaky expression of this inducible expression system. Moreover, we found that the tetracycline-controlled transcriptional silencer (tTS) does not effectively reduce the background expression of the Tet-On 3G system in sf9 cells. However, Csy4, a Cas9 homologous protein in the CRISPR family with sequence-specific endonuclease activity, was found to be effective in reducing the Tet-On 3G system-associated background expression, although there was a concomitant reduction in the maximum induced expression. Nevertheless, we found that modification of the system via incorporation of TRE-controlled anti-sense csy4 in combination with a WSSVie1 (Δ23) promotor-driven sense csy4 significantly reduced the leaky expression of the Tet-On 3G system, and that the level of induction was higher than that initially obtained. This optimized Tet-On 3G system can significantly reduce cell death attributed to the background expression of Kid under uninduced conditions. Therefore, we developed a novel low-background inducible expression system for use in insect cells and potentially in other organisms including mammals based on post-transcriptional regulation using Csy4.
Highlights
The tetracycline regulatory expression system is widely used to regulate gene expression and has been demonstrated to function in yeast [1], plants [2], insects [3, 4], and mammals [5, 6]
It has been reported that the Kid toxin can induce apoptosis in mammalian cells [14], and we examined whether Kid is toxic to insect cells
We found the Kid protein to be highly toxic to insect sf9 cells, and when we attempted to express Kid using the Tet-On 3G system, we observed that leaky expression of the Kid protein was sufficient to kill a proportion of the sf9 cells in the absence of the induction agent doxycycline
Summary
The tetracycline regulatory expression system is widely used to regulate gene expression and has been demonstrated to function in yeast [1], plants [2], insects [3, 4], and mammals [5, 6]. Owing to the detrimental effects of Tet-On system-associated background expression, considerable attention has focused on measures that can be adopted to reduce such unwanted expression; for example, by screening rtTA protein mutants that are more sensitive to doxycycline via viral evolution, and achieving higher induction efficacy using lower concentrations of doxycycline [7]. By optimizing the TRE promoter, Loew et al (2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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