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L.H. Pinto, M.H. Vitaterna, K. Shimomura, S.M. Siepka, V.C. Sheffield, E.M. Stone, R.F. Mullins, J.S. Takahashi; Molecular Cloning of the Noerg–1 Mutation in Mouse Shows Mutagenesis Can Generate Valid Disease Models . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3189.
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Purpose: The dominant Noerg–1 mutation was generated by chemical mutagenesis and causes loss of the ONL in 12 week old mice. We wished to identify the gene mutation responsible for the loss of photoreceptors. Methods: At 8–12 weeks of age the dark– adapted electroretinogram of 28 G5 mice was measured and quantified, the fundus was photographed and analyzed for abnormalities and the retina was examined with immunofluorescence and TUNEL analysis. Over 150 mutant offspring of mapping crosses with the DBA/2J and BALB/c strains were analyzed with informative SNP and SSLP markers and candidate genes in the nonrecombinant region were sequenced. Results: Histological and TUNEL analyses suggested that the photoreceptor cells die by apoptosis. The mutation mapped to Chromosome 6, distal to D6Mit287 (112.8 MB, 2/153 recombinants) and proximal of mCV22289734 (115.2 Mb, 1/153 recombinants). No recombinants were detected with a SNP marker within the rhodopsin gene. Genes falling within this region that are expressed in SAGE expression libraries include Rho GTPase binding protein (114.5 Mb), GATA4 (115.9 Mb), a GABA transporter 1 (116.0 Mb), rhodopsin (117 Mb) and ENSMUSG24104 (117.8 Mb). The sequence of the coding regions of each of these genes from the mutant was compared with that of wt and only one mutation was found. A missense mutation (A to G transition) causing a replacement of Cys110 of rhodopsin with Tyr was found in the mutant. Conclusions: Genetic analysis shows that the gene responsible for the phenotype is rhodopsin. Site–directed mutagenesis of this residue showed that elimination of this Cys residue results in improper folding of the molecule (PNAS 1999 vol. 96 p. 1932) and we speculate that this improper folding could lead to a dominant negative cell death. It is important to note that this very same amino acid substitution in humans results in autosomal dominant retinitis pigmentosa and this study thus shows that ENU mutagenesis is capable of producing authentic models for human disease.
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