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GD Aguirre, S Pearce-Kelling, GM Acland, Q Zhang; Retinal RPGR Expression in Normal Dogs, and in Mutants (XLPRA1/XLPRA2) Affected with Microdeletions of RPGR Exon ORF15 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3668.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Different microdeletions of canine RPGR exon ORF15 result in either late- or early-onset inherited retinal degeneration (XLPRA1, XLPRA2 respectively). To study the mechanisms of these diseases, we examined RPGR expression in normal and mutant retinas at different ages and disease stages. Methods: Western analysis of RPGR in cytosolic and membrane fractions used antibodies RPGR-254, 1878, and DR-39 which target different RPGR epitopes. Northern analysis used probes corresponding to different exons of RPGR cDNA. For in situ hybridization an exon ORF15 RNA probe was used. For immunocytochemistry, antibodies to RPGR and other photoreceptor-specific proteins were used. Results: Northerns with probes for exons 3-10 or ORF15 showed 2 major high molecular size retinal transcripts (8.9 and 5.0 kb). The 5 kb transcript, which is predominantly expressed in retina, matches the length (5284 bp) expected if this transcript splices in ORF15 as an alternative terminal exon, and skips exons 16 to 19 (present in the 8.9 kb band). Analysis of pre-degenerate mutant retinas showed both the 8.9 and 5 kb bands, with slightly lower levels of expression in XLPRA2 retinas. In situ hybridization showed intense accumulation of label in the RPE, rod and cone inner segments, and ONL/INL, that was similar in normal and XLPRA1. Westerns showed RPGR expression in normal and mutant retinas, but no differences in band sizes between them, an indication that the antibodies were not specific for ORF15 sequences; RPGR, opsin, PDEg, and the rod a channel protein were expressed normally by immunocytochemistry. Conclusion: Retinal expression of RPGR is complex, with multiple splice variants; in the retina-predominant transcript ORF15 is the terminal exon. RPGR is expressed in the retina of XLPRA1- and XLPRA2-mutant dogs. The mutations do not impair the expression and distribution of other photoreceptor-specific proteins. Support: EY06855, EY13132, Foundation Fighting Blindness, Van Sloun Fund for Canine Genetic Research
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