title: Mislocalization and degradation of human P23H-rhodopsin-GFP in a knockin mouse model of retinitis pigmentosa.
abstract: OBJECTIVE
  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.
authors: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors: Brandee A
authors: BA
authors: Price
authors: Ivette M
authors: IM
authors: Sandoval
authors: Fung
authors: F
authors: Chan
authors: David L
authors: DL
authors: Simons
authors: Samuel M
authors: SM
authors: Wu
authors: Theodore G
authors: TG
authors: Wensel
authors: John H
authors: JH
authors: Wilson
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D003062:Codon; D004195:Disease Models, Animal; D004596:Electroretinography; D005786:Gene Expression Regulation / Q000502:physiology; D055879:Gene Knock-In Techniques; D060005:Genotyping Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011392:Proline / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D055211:Retinal Photoreceptor Cell Inner Segment / Q000378:metabolism; D055214:Retinal Photoreceptor Cell Outer Segment / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012243:Rhodopsin / Q000235:genetics*
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D012243:Rhodopsin; D011392:Proline