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I. Sandoval, B.D. Gerwin, S. Boye, W.W. Hauswirth, T.G. Wensel, T.W. Kraft; AAV–Directed Overexpression of R9AP and RGS9–1 in Rat Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):853.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether expression of the RGS9–1 membrane anchor protein, R9AP, using a viral vector, can elevate levels of the RGS9–1 GAP complex in photoreceptors in order to test the effects of such elevation on photoresponse recovery kinetics and cell survival in degenerating retinas.
A recombinant serotype 5 adeno–associated virus (rAAV) construct directing expression of both R9AP and EGFP under control of rhodopsin promoters was subretinally injected in 6 week–old Sprague Dawley rats. The retinas were harvested 8–10 weeks later and used for quantitative immunoblotting. Retina samples from control and injected eyes of three different animals were homogenized in detergent and the soluble fraction was isolated by centrifugation. The proteins were separated with 12% SDS–PAGE gel electrophoresis and transferred to nitrocellulose membranes. Antibodies specific for RGS9, R9AP and rhodopsin were used to probe the membranes, and detected by chemiluminescence. Densitometry was used to calculate amounts of each protein in the sample using serial dilution of standards from bovine ROS (rod outer segments). R9AP and RGS9–1 levels were determined as ratios to levels of rhodopsin.
Fundus photographs of injected animals and isolated retinas showed rod cells positive for GFP expression near the site of injection. The extent of expression varied with the sample (1.6–2.6mm diameter patches). The samples positive for GFP showed over–expression of R9AP protein 25–30 fold higher than control samples. These large increases in R9AP were accompanied by more modest increases in RGS9–1 levels of 1.5–4.5 fold compared to the control samples.
Although the amount of expression varies from sample to sample, the AAV construct can efficiently direct expression of high levels of GFP and R9AP in rod cells under control of the rhodopsin promoter. The over expression of R9AP is sufficient to drive significant increases in RGS9–1 levels.
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