Retinal Degeneration Induced by Targeted Disruption of the Mouse Rp1 Gene

Abstract

Abstract: : Purpose:Mutations in photoreceptor gene RP1 account for 6-10% of autosomal dominant retinitis pigmentosa. All pathologic mutations in RP1 found to date result in premature termination of translation in the N-terminal half of the protein. Recently, we found that the RP1 protein is specifically localized to the connecting cilia of rod and cone photoreceptors (IOVS, Jan 2002). To help elucidate the function of RP1 in vision and the mechanism by which mutations in RP1 lead to photoreceptor cell death, we have generated a mouse model of the RP1 form of adRP by targeted disruption of the mouse Rp1 gene. Methods:The mouse Rp1 gene was disrupted by truncating the coding sequence after codon 665 (corresponding to the location of the most common human mutation Arg677Ter). A 10 amino acid myc tag was added to the 3' end of the truncated Rp1 coding sequence. Chimeric mice were generated from targeted mouse embyronic stem cells using standard techniques, and bred to produce mice that are heterozygous (Rp1_+/myc) or homozygous (Rp1_myc/myc) for the mutant Rp1-myc allele. RT-PCR, Western blotting and immunostaining were performed to detect expression of the mutant Rp1-myc protein in the retina. Retinal morphology and function in the mutant Rp1-myc mice and littermate controls were assessed by light and electron microscopy and electroretinograms. Results:Southern blotting and PCR analyses demonstrate that the Rp1 locus has been successfully targeted with the mutant allele. Retinas from Rp1_+/myc mice demonstrated expression of both the normal Rp1 and the truncated Rp1-myc proteins the in connecting cilia of photoreceptor cells. Only mutant Rp1-myc protein was detected in retinas from Rp1_myc/myc. By 4 weeks of age, retinas in Rp1_myc/myc mice showed significant degenerative changes. The thickness of outer nuclear layer and the outer-segment length were reduced by approximately 30% compared to wild-type littermate controls. The rod ERG response in 4-week old Rp1_myc/myc mice was reduced by approximately 50%. Evaluation of retinas from Rp1_+/myc mice is in progress. Conclusion:The mutant Rp1-myc allele in these mice produces a truncated Rp1 mRNA and protein. This implies that patients with mutations in RP1 may make truncated versions of the RP1 protein in their retinas. If this is true, mutations in RP1 may cause photoreceptor degeneration via a dominant negative effect of the truncated RP1 proteins in the connecting cilia of photoreceptor cells. Further evaluation of the heterozygous Rp1_+/myc animals will help clarify this issue, and provide insight into the pathogenesis of the RP1 form of adRP.

Keywords: 316 animal model