Tet-responsive Element Research Articles

Development of multigene and regulated lentivirus vectors.

Previously we described safe and efficient three-component human immunodeficiency virus type 1 (HIV-1)-based gene transfer systems for delivery of genes into nondividing cells (H. Mochizuki, J. P. Schwartz, K. Tanaka, R. O. Brady, and J. Reiser, J. Virol. 72:8873-8883, 1998). To apply such vectors in anti-HIV gene therapy strategies and to express multiple proteins in single target cells, we have engineered HIV-1 vectors for the concurrent expression of multiple transgenes. Single-gene vectors, bicistronic vectors, and multigene vectors expressing up to three exogenous genes under the control of two or three different transcriptional units, placed within the viral gag-pol coding region and/or the viral nef and env genes, were designed. The genes encoding the enhanced version of green fluorescent protein (EGFP), mouse heat-stable antigen (HSA), and bacterial neomycin phosphotransferase were used as models whose expression was detected by fluorescence-activated cell sorting, fluorescence microscopy, and G418 selection. Coexpression of these reporter genes in contact-inhibited primary human skin fibroblasts (HSFs) persisted for at least 6 weeks in culture. Coexpression of the HSA and EGFP reporter genes was also achieved following cotransduction of target cells using two separate lentivirus vectors encoding HSA and EGFP, respectively. For the regulated expression of transgenes, tetracycline (Tet)-regulatable lentivirus vectors encoding the reverse Tet transactivator (rtTA) and EGFP controlled by a Tet-responsive element (TRE) were constructed. A binary HIV-1-based vector system consisting of a lentivirus encoding rtTA and a second lentivirus harboring a TRE driving the EGFP reporter gene was also designed. Doxycycline-modulated expression of the EGFP transgene was confirmed in transduced primary HSFs. These versatile vectors can potentially be used in a wide range of gene therapy applications.

Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system.

Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege.

Expression of Green Fluorescent Protein in Oligodendrocytes in a Time- and Level-Controllable Fashion with a Tetracycline-Regulated System

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.

Altered Oxygen Toxicity with Variable Overexpression of Heme Oxygenase-1 in Hamster Fibroblasts ♦ 352

Heme oxygenase (HO) is the rate limiting enzyme in bilirubin production. The HO-1 isoform is inducible by a variety of oxidant stresses including hyperoxia(1). In its reaction, heme, a prooxidant is degraded, bilirubin, an antioxidant is formed(2). Thus, the overall effect of HO may be to protect against oxidative stress. Previous results from rat fibroblasts transfected with an expression vector containing the rat HO-1 cDNA have led us to speculate that there may be a beneficial threshold of HO-1 overexpression. To further investigate this hypothesis, we have established a variable overexpression model of HO-1 using the Tet-Off™ Gene Expression System (Clontech) which is based on the tetracycline resistance system found in E. coli(3). In this system, gene expression is controlled by the interaction of a tet-responsive transcriptional activator protein and a tet-responsive element where gene expression is tightly regulated by varying concentrations of either tetracycline or doxycycline (Dox) in a dose dependent manner. After insertion of the full length rat HO-1 cDNA into the pBI-L response plasmid, two consecutive stable transfections were performed and >60 stable colonies were screened in order to find a cell line allowing for regulated variable HO-1 overexpression. This cell line was grown in 75cm flasks to >80% confluence. To verify HO-1 overexpression, total HO activity was measured by gas chromatography and HO-1 protein was assessed by Western analysis. By varying the concentration of Dox between 0 and 10ng/ml, we were able to regulate HO-1 expression between 1.5 and 20 fold. Once confluent, cell lines expressing a range of HO-1 protein were exposed to hyperoxia (95% O2/5% CO2) for 72 h and non-specific indicators of cell injury were measured by Trypan blue exclusion and LDH release. Lipid peroxidation, protein oxidation, and total glutathione were also evaluated as specific markers of oxygen toxicity. Lower HO-1 overexpression was associated with increased protection against oxygen damage; however, higher expression was associated with increased markers of cell injury. In this model, we conclude that there is a beneficial threshold of HO-1 overexpression beyond which detrimental effects become apparent. We suggest that measures to enhance protection against oxygen toxicity by HO-1 overexpression be undertaken with a thorough understanding of the potential dual nature of HO-1.

Integration of Tetracycline Regulation into a Cell-specific Transcriptional Enhancer

The pancreas-specific transcriptional enhancer of the rat elastase I gene was modified by substituting, in turn, each of its three individual constitutive elements with the tetO element, which confers regulation by exogenous tetracycline in the presence of the hybrid tetO binding transactivator (tTA). Whereas the unmodified enhancer was active in transfected acinar tumor cells, substitution of individual elements with the tet-responsive element abolished activity. The modified enhancers were reactivated in the presence of the tTA and, upon addition of tetracycline, were silenced. Thus, substitution of individual enhancer elements renders the enhancer responsive to regulation by tetracycline. Moreover, the tTA-activated levels were 2-8-fold greater than the unmodified enhancer. The acinar cell specificity of the unmodified enhancer was retained; none of the tetO-substituted enhancers were activated by tTA in a variety of nonacinar cell lines. These results show that a foreign and artificial transcriptional activator, tTA, can be incorporated into an enhancer to create a novel, efficient, and regulatable transcriptional control region whose cell specificity is retained.