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S.X. He, H. Khanna, C. Lillo, G. Acland, S. Pearce–Kelling, G. Aguirre, D. Williams, A. Swaroop; RPGR isoforms in the retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2437.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations in Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the primary cause of X–Linked retinitis pigmentosa. RPGR is ubiquitously expressed, with several distinct isoforms including predominantly retina–specific RPGR–ORF15. Various studies have reported different localization(s) of RPGR in the retina. The aims of the present study are to generate a panel of antibodies directed against RPGR and characterize the different isoforms expressed in the retina. Methods: Four peptide antibodies were generated based on the deduced amino–acid sequence of RPGR–ORF15. These antibodies were tested in an immunoblot assay using human, mouse, bovine and canine retinal protein extracts. Subcellular fractionation of bovine retinal extracts was performed by differential centrifugation. Results: To specifically evaluate the RPGR–ORF15 isoform(s), immunoblots of retinal extracts from the Rpgr–knockout mouse (Hong et al. PNAS 2000) and the XLPRA1 dog (Zhang et al. HMG 2002) were analyzed. One of the antibodies generated against the C–terminus (hORF15c) identified a major 200 kDa band in the wild type mouse retina and 3–4 bands of 150–200 kDa in the bovine and normal dog retina. These proteins were not detected in the Rpgr–knockout or the XLPRA1 dog retinal extracts. Immunogold microscopy using the hORF15c antibody showed that Rpgr is primarily localized in the connecting cilium of photoreceptors though the protein is also detected in the inner and outer segments. Subcellular fractionation of bovine retina extracts demonstrated that the 200 kDa band was present in the cytosol, whereas the smaller isoforms of around 150 kDa were observed in the nuclear fraction. Additional studies are in progress. Conclusions: Our data confirm the presence of several RPGR isoforms with distinct localization(s) in the retina. It is conceivable that these isoforms have discrete functions, which contribute to the disease phenotype.
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