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How to make tetracycline-regulated transgene expression go on and off
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Vascular Biology)
2002 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 309, no 1, 79-84 p.Article in journal (Refereed) Published
Abstract [en]

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

Place, publisher, year, edition, pages
2002. Vol. 309, no 1, 79-84 p.
Keyword [en]
Doxycycline, Transgene, Gene therapy, Regulatable, tTA-responsive promoter
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-91471DOI: 10.1016/S0003-2697(02)00250-6PubMedID: 12381365OAI: oai:DiVA.org:uu-91471DiVA: diva2:164210
Available from: 2004-03-09 Created: 2004-03-09 Last updated: 2011-06-28Bibliographically approved
In thesis
1. Molecular Mechanisms in Endothelial Cell Differentiation
Open this publication in new window or tab >>Molecular Mechanisms in Endothelial Cell Differentiation
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays.

The role of the signaling protein H-Ras in FGF-2-induced in vitro angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. In vitro angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation.

In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 34 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1328
Pathology, angiogenesis, FGF-2, VEGF-A, signal transduction, in vitro angiogenesis assay, immortalized brain endothelial cells, H-Ras, tetracycline-regulated gene expression, microarray, telomerase immortalized human microvascular endothelial cells, differentiation, Patologi
National Category
Cell and Molecular Biology
urn:nbn:se:uu:diva-4059 (URN)91-554-5900-5 (ISBN)
Public defence
2004-04-02, Rudbeckssalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15
Available from: 2004-03-09 Created: 2004-03-09Bibliographically approved

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Gerwins, Pär
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