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W. Li, B. Janic, X. Zhang; Lentivirus-Mediated Gene Transfer in Retinal Stem Cells - Effects of Long-Term Expression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4090.
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Retinal stem cells (RSCs)/progenitors are critical for the development of therapeutic approaches for degenerative retinal diseases. Lentivirus-mediated gene transfer is an important strategy for RSC ex vivo modulation. This study is to investigate long-term gene expression mediated by lentiviral vector in RSCs.
RSCs were isolated from mouse retina at postnatal day 1 and cultured in serum-free culture medium supplemented with EGF and FGF. Retinal spheres were transduced with GFP-expressing feline immunodeficiency virus (FIV). GFP expression was analyzed by flow cytometry and fluorescent microscope. RSCs were differentiated in mitogen-free culture medium supplemented with 5% FBS. Expression of cell-specific markers was analyzed by immunocytochemistry in both undifferentiated and differentiated RSCs. Differentiated postmitotic cells were revealed by BrdU incorporation assay.
Stem cell marker nestin was detected in the cultured retinal spheres. FIV transduced more than 97% of retinal spheres with sustainable GFP expression for at least two months in culture. Upon differentiation, cell-specific markers, including MAP-2, opsin and GFAP, were detected in FIV-transduced or control RSCs at similar levels, suggesting that FIV transduction did not affect cell differentiation. However, GFP expression substantially decreased in BrdU-negative differentiated cells, suggesting the downregulation of the transgene expression in differentiated postmitotic cells.
FIV-mediated gene transfer induces sustainable gene expression in undifferentiated RSCs. However, the transgene expression is down-regulated in differentiated RSCs.
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