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  • Viral Transfer Technology
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Adenoviral vector which delivers FasL–GFP fusion protein regulated by the tet-inducible expression system

Gene Therapy volume 7, pages 875–885 (2000)Cite this article

Abstract

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.

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Acknowledgements

We wish to thank Dr James Norris (Department of Microbiology and Immunology, Medical University of South Carolina) for his advice and assistance in the preparation of this manuscript. This study was supported in part by Grants for American Cystic Fibrosis Foundation: DONG96PO, DONG97PO and Grants from NIH: DK46177 and DK53217.

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  1. S Rubinchik & J Dong

    Present address: Departments of Microbiology and Immunology, and Medicine, Medical University of South Carolina, Charleston, SC, USA

Authors and Affiliations

  1. Department of Laboratory Medicine, University of California, San Francisco, California, USA

    S Rubinchik, R Ding, A J Qiu & J Dong

  2. Department of Ophthalmology, University of California, San Francisco, California, USA

    F Zhang

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Rubinchik, S., Ding, R., Qiu, A. et al. Adenoviral vector which delivers FasL–GFP fusion protein regulated by the tet-inducible expression system. Gene Ther 7, 875–885 (2000). https://doi.org/10.1038/sj.gt.3301172

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