Clarin 1 Is Necessary for Hearing but Not Vision Figure 6. Histological analysis of Clrn1 KO retinas at 9 months of age. (A–C) Compared to WT animals (A), outer retinal histology appears normal in heterozygous (B) and homozygous (C) Clrn1 KO mice. (D–F) Similarly, the inner retina of the Clrn1 KO (+/2,2/2) animals is indistinguishable from the WT animal (D). (G–L) When compared to WT (+/+) animals (G and J) at lower magnification, gross retinal structure seems unperturbed by the lack of Clrn1 expression (H,I,K,L) in either mid-peripheral retina (G–I) or central retina (J–L) immediately adjacent to the optic nerve head (asterisks). Scale bar = 50 mm. ONL = outer nuclear layer, IS = photoreceptor inner segments, OS = photoreceptor outer segments, and RPE = retinal pigmented epithelium. doi:10.1371/journal.pgen.1000607.g006 (Figure S4), driven by the human cytomegalovirus (CMV) immediate early promoter. Transfection with the vector alone resulted in EGFP expression localized throughout cell somata (Figure S4A). CLRN1 (containing exons 1, 3, and 4) was fused to the 39 end of EGFP, and following expression the resultant fusion- protein was redistributed and concentrated to plasma membranes (Figure S4B). This finding supports previous predictions based upon the primary structure[12], as well and recent studies [23], that full-length murine CLRN1 (232 aa) is predominantly a plasma membrane-targeted protein. Anti-CLRN1 antibodies were screened for specificity using EGFP-CLRN1 transfections in HEK293 cells. With the red channel turned off, clear EGFP-CLRN1 fluorescence is observed in a subset of cells (Figure S4C). Red fluorescence (Figure S4D) PLoS Genetics | www.plosgenetics.org 7 August 2009 | Volume 5 | Issue 8 | e1000607