Mislocalization and degradation of human P23H-rhodopsin-GFP in a knockin mouse model of retinitis pigmentosa

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PURPOSE: To engineer a knockin mouse model that can be used to monitor the effects of treatments on degradation and mislocalization of proline-to-histidine change at codon 23 (P23H) rhodopsin, a common cause of autosomal dominant retinitis pigmentosa (ADRP). The goal was to introduce a gene that expressed rhodopsin at low levels to avoid rapid retinal degeneration, and with a readily visible tag to make it easy to distinguish from wild type rhodopsin. METHODS: One copy of the endogenous mouse rhodopsin gene was replaced with a mutant human rhodopsin gene that encodes P23H-rhodopsin fused to enhanced green fluorescent protein (GFP) at its C terminus. The gene includes a LoxP site in the sequence corresponding to the 5'-untranslated region, which greatly reduces translation efficiency. Characterized are the resulting heterozygous and homozygous P23H-hRho-GFP mouse lines for mRNA and protein expression, P23H-rhodopsin localization in rod cells, effects on visual function, and retinal degeneration. RESULTS: The retinas of heterozygous P23H-hRho-GFP mice are morphologically and functionally very similar to those of wild type mice, and they display little cell death over time. P23H-hRho-GFP mice transcribe the knockin gene as efficiently as the endogenous mouse allele, but they contain much less of the protein product than do knockin mice expressing nonmutant hRho-GFP, indicating that substantial degradation of P23H-rRho-GFP occurs in mouse rod cells. The remaining P23H-hRho-GFP mislocalizes to the inner segment and outer nuclear layer, with only approximately 20% in rod outer segments. CONCLUSIONS: P23H-hRho-GFP mice provide a valuable tool for evaluating the efficacy of potential therapies for ADRP that influence the levels or localization of P23H-rhodopsin.

Medical Subject Headings

Animals; Blotting, Northern; Codon; Disease Models, Animal; Electroretinography; Gene Expression Regulation (physiology); Gene Knock-In Techniques; Genotyping Techniques; Green Fluorescent Proteins (genetics); Histidine (genetics); Mice; Mice, Inbred C57BL; Microscopy, Confocal; Mutagenesis, Site-Directed; Mutation; Proline (genetics); Recombinant Fusion Proteins (genetics); Retinal Photoreceptor Cell Inner Segment (metabolism); Retinal Photoreceptor Cell Outer Segment (metabolism); Retinitis Pigmentosa (genetics, metabolism, pathology); Rhodopsin (genetics)

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Investigative ophthalmology & visual science







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