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Brandee A. Price, Ivette M. Sandoval, Fung Chan, David L. Simons, Samuel M. Wu, Theodore G. Wensel, John H. Wilson; Mislocalization and Degradation of Human P23H-Rhodopsin-GFP in a Knockin Mouse Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2011;52(13):9728-9736. doi: 10.1167/iovs.11-8654.
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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.
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.
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.
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.
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