Rubinchik, S, Woraratanadharm, J, Yu, H and Dong, JY. New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo. Gene Ther 12: 504-511

Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29403, USA.
Gene Therapy (Impact Factor: 3.1). 04/2005; 12(6):504-11. DOI: 10.1038/
Source: PubMed


Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.

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Available from: Hong Yu, Oct 09, 2014
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    • "tTS Kid totally abrogated basal transgene leak in the absence of doxycycline , without impairing the ability of this inducer to stimulate rtTA-mediated IL-13 expression. To optimize delivery of this activation/repression system, all components were incorporated in single adenoviral or episomally replicating vectors that performed well both in vitro and in vivo (Salucci et al., 2002; Mizuguchi et al., 2003; Bornkamm et al., 2005: Rubinchik et al., 2005; Xiong et al., 2006). However, inclusion in tetracycline-regulated gene switches of tTS was also reported to result in reduced activation of transgenes (Lamartina et al., 2003). "

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    • "So far, several kinds of vectors have been used to construct Tet or Dox-inducible expression systems such as lentiviral [5], [6], retroviral [7], adeno-associated viral [1], [2], [8]–[11], first-generation adenoviral [2], [9], [10], herpesviral [12], high-capacity adenoviral [13]–[18] and the bacterial artificial chromosome (BAC) vectors [19]. BAC vectors are optimal for harboring relatively long fragments of genomic DNA or large cDNA in excess of 100 kb. "
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    ABSTRACT: Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.
    Full-text · Article · Feb 2009 · PLoS ONE
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    • "Although some tTS' enabled tetracycline controlled gene expression from appropriately engineered promoters in mammalian cells (Deuschle et al. 1995), they did not became popular as independent regulatory systems. Instead they proved very useful in combination with the Tet-on systems, reducing residual leakiness in the absence of induction (Rossi et al. 1998; Forster et al. 1999; Zhu et al. 2001; Lamartina et al. 2003; Mizuguchi et al. 2003; Rubinchik et al. 2005). The most commonly used construct of this type is the fusion of a mutant TetR and the KRAB-AB domain of the Kid-1 protein (Freundlieb et al. 1999). "
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    ABSTRACT: The increasing number of proteomic and DNA-microarray studies is continually providing a steady acquisition of data on the molecular abnormalities associated with human tumors. Rapid translation of this accumulating biological information into better diagnostics and more effective cancer therapeutics in the clinic depends on the use of robust function-testing strategies. Such strategies should allow identification of molecular lesions that are essential for the maintenance of the transformed phenotype and enable validation of potential drug-targets. The tetracycline regulated gene expression/ suppression systems (Tet-systems) developed and optimized by bioengineers over recent years seem to be very well suited for the function-testing purposes in cancer research. We review the history and latest improvements in Tet-technology in the context of functional oncogenomics.
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