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I.M. MacDonald, C.–C. Chan, K.T. Hiriyanna, D. Shen, R. Fariss; REP–1 Localization in the Eye . Invest. Ophthalmol. Vis. Sci. 2005;46(13):540.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations in the CHM gene are responsible for the X–linked retinal degeneration choroideremia. This gene encodes Rab escort protein–1 (REP–1) which is expressed in the retina and is believed to play a critical role in intracellular protein targeting. In this study, the expression and subcellular localization of the normal CHM gene product, REP–1, were evaluated in a variety of mammalian species. These studies of REP–1 expression in normal retina provide a basis for understanding this protein's contribution to normal retinal function as well as the retinal cell types impacted in patients with choroideremia. Methods: Immunohistochemistry: Antibodies to REP–1 were generated (2F1 mouse monoclonal, 09K rabbit polyclonal) and used for immunolabeling studies in paraffin and frozen sections of eyes from mouse, rat, monkey and human. A blocking peptide was used to verify specificity of the 09K ployclonal REP–1 antibody in immunolabeling studies. Confocal microscopy was used to evaluate the subcellular distribution of REP–1 in cryosections. Microdissection and RT–PCR: Photoreceptor and RPE cells were microdissected from normal human and monkey retinal frozen sections. RNA was extracted and isolated for the evaluation of CHM message using RT–PCR with primer pairs of CHM–281 (AAG CCA TTG CTC TTA GCA GG) and CHM–532 (CTA GCG CAT TCT CTG GAT CG). Results: Immunolabeling studies demonstrated that REP–1 protein was expressed throughout the retina, including rod and cone photoreceptors. In rods and cones labeling was prominent in inner segments and the perinuclear cytoplasm in each of the four mammalian species examined in this study. The addition of blocking peptide completely abolished immunolabeling. Microdissection and RT–PCR analysis confirmed the presence of CHM message in these neurons. Conclusions: REP–1 is expressed in both rod and cone photoreceptors in a variety of mammalian species, including human and non–human primates. Mutations in REP–1 may compromise the viability of photoreceptors by interfering with protein targeting in these highly compartmentalized neurons.
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