Tetracycline-regulatable expression vectors tightly regulate in vitro gene expression of secreted proteins |
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Affiliation: | 1. Progenitor Inc. 4040 Campbell Avenue, Menlo Park, CA 94025, USA;2. Edison Biotechnology Center, Ohio University, Athens, OH 45701, USA;3. Ontogeny Inc., 45 Moulton St., Cambridge, MA 02138, USA;1. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands;2. Department of Cardiothoracic Surgery, Leiden University Medical Center, Leiden, the Netherlands;1. Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), 07745 Jena, Germany;2. Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, 07745 Jena, Germany |
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Abstract: | The regulation of gene expression by the tetracycline system has attracted a high level of interest in the recent past. However, expression of secreted proteins has not been evaluated precisely. In this study, we constructed two versions of a one-plasmid system containing the elements necessary for the regulation of gene expression. The regulatable elements and the selectable marker (Neor) were set up in two different configurations, pTRIN31 and pTRIN76. With these two regulatable versions, the levels of protein expression after transfection into the NIH/3T3 cell line were measured by insertion of three different genes encoding the secreted proteins (hGH, ApoE3, hGM-CSF). The maximum levels of gene expression obtained with the pTRIN76-derived plasmids were 100 ng/24 h/106 cells for hGH, 427 ng/24 h/106 cells for ApoE3 and 108 ng/24 h/106 cells for hGM-CSF. For the pTRIN31-derived plasmids the maximum levels were 2.7 ng/24 h/106 cells for hGH and 47 ng/24 h/106 for ApoE3. Both plasmids give rise to an expression of the transfected gene that can be tightly regulated by three different molecules: tetracycline, minocycline and doxycycline. The levels of the secreted proteins are below the detectable level when the reporter genes are repressed. This repression is reversible within 48 h after the regulator has been removed from the medium. |
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