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S.F. Geller, E. Sankila, A. Adato, J.G. Flannery; Clarin-1 Expression in the Mouse Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2270.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To further investigate the expression of Clarin-1, the product of the gene responsible for Usher Syndrome type 3, in the mouse retina. Methods: Four antibodies were made in New Zealand White rabbits (Sigma) to two synthetic polypeptides. One antibody (Clarin1B2) was subsequently affinity purified (with the peptide) and used in immunohistochemical, immunoprecipitation and Western analyses. Western analysis was performed on human, mouse and rat retinal homogenates. RT-PCR analysis of the entire Clarin-1 coding region was performed on mouse total retinal RNA. Results: Immunohistochemistry using the purified antibody localized Clarin-1 specifically to horizontal cells in mouse retina, but failed to reproducibly label in either rat or human retina. At higher concentrations, the antibody also labels Müller cells in all retinas tested. Western analysis shows specific immunoreactivity with a ~26kDa band on a SDS-PAGE reducing gel, precisely the molecular weight of the primary Clarin-1 transcript (230 amino acids; excluding exon 1). After immunoprecipitation and Western blotting on a non-denaturing (no SDS) gel, Clarin1B2 identified a protein (or protein complex) of approximately 105kDa. RT-PCR analysis suggested two transcripts are expressed in the mouse retina; one "full length" transcript (excluding exon 1), and a similar transcript containing a presumed splice variant in exon 3. This transcript lacks 28 nucleotides, and thus, alters the reading frame and creates a novel stop codon 4 amino acids later. Furthermore, this small deletion truncates the full-length protein by 37 amino acids. Conclusions: An antibody to Clarin-1 shows labeling of horizontal cells in the mouse retina. Western blotting confirms immunoreactivity with a ~26kDa protein, and immunoprecipitation experiments suggest that Clarin-1 may multimerize or be part of a larger protein complex. RT-PCR analysis confirms the expression of full-length Clarin-1 (excluding exon 1) in the mouse retina, previously found in the mouse inner ear. In addition, a novel transcript coding for a truncated Clarin-1 transcript was found. Although only a paucity of data exists on this newly identified gene, steps are being made to elucidate the localization, expression, and function of Clarin-1 in health and disease.
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