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K.P. Greenberg, E.S. Lee, J.N. Leonard, D.V. Schaffer, J.G. Flannery; Temporal Tropism for Recombinant Lentiviral-GFP Transduction of Photoreceptors in C57BL/6 Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):450.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies using lentiviral vectors to deliver green fluorescent protein (GFP) to the mouse and rat retina via subretinal injection have shown transduction and subsequent expression in the retinal pigment epithelium and photoreceptors. This investigation demonstrates a specific developmental window for lentiviral transduction and expression of GFP in photoreceptors of the mouse retina. Methods: Recombinant lentivirus pseudotyped with vesicular stomatitus virus glycoprotein (VSVG) carrying a GFP reporter gene under the control of the CMV promoter was packaged through the calcium phosphate transient transfection of four plasmids into 293T cells. High titer viral stocks of 109 infectious units/ml were obtained by ultracentrifugal concentration of the cell supernatant. Young C57BL/6 mice (age P4-P17) were injected subretinally with 2.5x105-2x106 infectious units and sacrificed ten days post injection, at which point the expression pattern of GFP was analyzed by fluorescent microscopy of retinal cryosections. Results: Retinas of mice injected prior to P7 showed localized expression of GFP in photoreceptors primarily near the injection site. Mature retinas however, did not contain photoreceptors visibly expressing GFP. Retinas of all mice injected in this study, regardless of age or number of viral particles delivered, exhibited strong expression of GFP in the retinal pigment epithelium. Conclusion: These results indicate a precise developmental window for VSVG pseudotyped lentiviral transduction of photoreceptors in C57BL/6 mice, after which only the RPE is readily transduced by this viral construct.
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