Abstract
BackgroundOne of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.ResultsWe constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.ConclusionsOur novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.
Highlights
One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes
By utilizing a tetracycline-controlled transcriptional silencer and the latest version of reverse Tet transactivator (rtTA), we constructed a single adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop in the E1 region to improve the regulational properties of the Ad-mediated Tet-On system. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand in SW480 cells and its tumor-derived cells inhibitory activity under induced conditions
Generation of a novel adenoviral E1 shuttle-based, all-in-one Tet-On system with an autoregulatory loop The major problem with an adenoviral vector carrying a Tet-On system is the suboptimal regulation of transgene expression
Summary
One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression. The adenovirus (Ad) vector is one of the most commonly used vectors for gene therapy. The tightly regulated expression of transgenes by adenoviruses is very important for optimizing therapeutic outcomes. In the Tet-Off system, by using the tetracycline transactivator (tTA) protein, transcription is turned off in the presence of tetracycline. In the Tet-On system, by using the reverse Tet transactivator (rtTA) fusion protein, transcription is turned on in the presence of tetracycline.
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