Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

Elise Orhan,Christophe Lechauve,Thierry Léveillard,Christina Zeitz,Christelle Michiels,Matthew M Lavail,Deniz Dalkara,Serge Picaud,José-Alain Sahel,Muna I Naash,Isabelle Audo,Marion Neuillé

doi:10.1371/journal.pone.0127319

Abstract

Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.

Highlights

Rod-cone dystrophy, known as retinitis pigmentosa (RP), is a clinically and genetically heterogeneous group of progressive inherited retinal disorders, which often start with night blindness and lead to visual field constriction, secondary macular involvement and in many cases results in loss of central vision and complete blindness [1]
We first investigated the transgene sequence of the P23H Line rat (P23H-1) rat model thought to be a mouse P23H mutated allele inserted in a WT rat background
Sanger sequencing with mouse Rho specific primers revealed that this transgene contains the entire mouse opsin (Rho) genomic DNA from the promoter to the 3’untranslated region (UTR) encompassing all exons and introns (Fig 1A)

Summary

Introduction

Rod-cone dystrophy, known as retinitis pigmentosa (RP), is a clinically and genetically heterogeneous group of progressive inherited retinal disorders, which often start with night blindness and lead to visual field constriction, secondary macular involvement and in many cases results in loss of central vision and complete blindness [1]. Mutations in the rhodopsin-encoding gene (RHO; MIM# 180380) were the first molecular defects identified in RP [2,3,4,5]. More than 100 different RHO mutations have been reported to date, which account for 30–40% of autosomal dominant RP (adRP) cases in United States [1] and 16% in France [6]. Uth.tmc.edu/Retnet/, last accessed date on December 16th, 2014) with mutations in RHO being the most prevalent [6, 7]. RHO encodes for the rod-specific protein rhodopsin, which belongs to the superfamily of 7 transmembrane G-protein—coupled receptors. The chromophore isomerizes into alltrans-retinal, with subsequent rhodopsin conformational changes leading to the activation of the phototransduction cascade [8]

Methods

Results

Conclusion