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L.G. Glushakova, A.M. Timmers, T.M. Issa, N.G. Cortez, J. Pang, W.W. Hauswirth; Does a Limited Portion of the Mouse Rod Opsin Promoter Support Only Rod Type–specific Expression in Rodents? . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3078.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Although a –385/+86 portion of the mouse rod opsin promoter (mOp500) delivered by an AAV vector has the advantage of exclusively targeting photoreceptors (PRs), the PR subtype specificity of the expression is yet unanalyzed. Here we evaluate whether this limited promoter region supports only rod specific expression or targets reporter expression to cones as well. Methods:Two µl of the purified AAV5.–mOp500.–GFP viral vector suspension ( 2.6*1013 vector genomes/ml) was administered subretinally to male Sprague–Dawley rats at PN40–48 and analyzed two weeks to eight months later. One µl of the same vector suspension was injected subretinally into rhodopsin–knockout (rho–/–) mice at PN18 and at PN78 and analyzed 12 days later. GFP–immuno– and PNA–lectin cytochemistry and morphological criteria were employed to characterize the patterns of GFP–reporter gene expression in PRs. Results:In rats, subretinal injection of rAAV5.–mOp500.–GFP vector generated abundant reporter gene expression in both rods and cones. GFP–positive cones, defined by morphological criteria and co–linearity of PNA–lectin labeling and GFP–immuno–reactivity, were found in all regions of the transduced retinal field. They constituted up to 5% of the total GFP–positive PRs (cones are 1% of the rat photoreceptors). By two weeks after administration, GFP labeling was found exclusively in the outer nuclear layer while after 8 months a low level of the GFP–reactivity was also observed in the inner nuclear and ganglion cell layers. In rho–/– mice, subretinal vector administration resulted in PR–exclusive GFP expression 12 days later. At PN30 immuno–cytochemical examination of PN18 treated eyes revealed that up to15% of the GFP–positive PR’s were cones (3% of mouse PR’s are cones). At PN90, eyes injected at PN78 had only a one single row of PR’s remaining, which was composed of the numerous PNA–labeled GFP–positive cones, most with severely degenerated outer segments. Conclusions:A limited portion (–385/+86) of the mouse opsin promoter shows a lack of the PR subtype–specificity in vivo in both, mouse and rat retinas. Thus this promoter is more correctly characterized as PR specific rather than rod specific. Relative to PR subtype specificity, it is interesting that similarly proximal human red– and blue–opsin promoter sequences support relatively specific cone expression with rod expression seen at 1,500 fold lower in transduction efficiency, (ARVO, 2003; 2004).
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