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D.M. Paskowitz, K.P. Greenberg, D. Yasumura, D. Grimm, M.A. Kay, M.M. LaVail, J.G. Flannery, D. Vollrath; Rapid and Stable Knockdown of the Endogenous Neuroprotective Factor bFGF in the Retina in vivo Using Viral Vectors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4826.
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The endogenous retinal expression of neuroprotective factors such as basic fibroblast growth factor (bFGF) has been proposed to promote photoreceptor survival during injury from inherited retinal degeneration or constant light exposure. To study the requirement for this factor and the cellular interactions that may contribute to photoreceptor survival, we used viral vector mediated expression of short hairpin RNAs (shRNAs) to knock down bFGF expression in vivo. Despite the promise of this approach for analyzing gene function in the retina, we are not aware of any published reports of the use of viral expression of shRNAs to knock down endogenous genes in the mammalian retina in vivo.
shRNAs were generated by PCR and tested in culture by transient transfection into HEK 293 cells, together with a bFGF–GFP fusion target. The expression level of the target was determined by RT–PCR and Western blotting. Successful shRNAs that demonstrated potent knockdown of the target were cloned into HIV–1 based lentiviral and double–stranded adeno–associated viral (dsAAV) vector backbones together with a GFP marker, and packaged into infectious particles by standard methods. Viral vectors were injected into the subretinal space of Sprague–Dawley albino rats. bFGF expression was studied 1–4 weeks later by immunostaining and colocalization with GFP.
Four shRNAs were identified that produced 75–90% knockdown of the bFGF–GFP target in cell culture. When expressed in vivo using either lentiviral or dsAAV–5 vectors, the most potent shRNA reduced expression of bFGF in RPE below the detection threshold of immunostaining. For both vectors, knockdown was evident by 1 week after injection and persisted for at least 4 weeks.
Viral expression of shRNAs from either of two different vectors produces rapid and stable knockdown of bFGF in retinal cells in vivo. This system can be used to test the requirement for this and other endogenous neurotrophic factors in retinal responses to stress.
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