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Zhaoxia Zhang, Henry KW Fong; Rgr gene mutation and exon-skipping RGR mRNA in mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4470.
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© ARVO (1962-2015); The Authors (2016-present)
Various abnormal RGR opsins lead to different retinal disease phenotypes. A rare human RGR mutation (c.196A>C, p.S66R) causes autosomal recessive retinitis pigmentosa, and another (c.824dupG, p.I276Nfs*77) leads to progressive peripapillary choroidal atrophy that is dominantly inherited. Our purpose is to study intracellular processing and biological effect of a mouse isoform of the human RGR-d exon-skipping protein in an animal model.
Multiple RGR-d mutant mouse lines (strains: B6D2F1/J and Rosa26-Cas9 knockin) were created by mutation of the splice site at each end of exon VI of the mouse Rgr gene. Destruction of the splice sites was performed by multiplex gene editing using CRISPR methods. A pair of single guide RNAs (gRNA), that targets the two splice sites of mouse Rgr exon VI, and Cas9 mRNA were microinjected into single-cell wildtype B6D2F1/J embryos. The pair of gRNAs alone was microinjected into embryos from Rosa26-Cas9 knockin mice.
We obtained mice with mutation of either the 5’ or 3’ splice site, both splice sites, or with total deletion of exon VI. We analyzed one litter of 8 viable pups from microinjection of wildtype B6D2F1/J embryos. The founders were designated F81, F82, F83, F89, M91, M94, F95, and F97. We obtained a litter of 10 pups from microinjection of Rosa26-Cas9 knockin embryos. Exon-VI-skipping RGR mRNA, or frameshift RGR transcripts, in positive F1 mice were confirmed by RT-PCR mRNA analysis and direct sequencing of the amplicons. Full-length RGR was completely absent on Western immunoblots of proteins from homozygous mutant (Rgr-d) mouse eyes, in which we found that the RGR-d isoform was expressed at very low levels (<1% of RGR in wildtype mice). At two weeks of age, little change was noted by light microscopy between the retinas of wildtype and Rgr-d F2 littermates. At five months of age, RPE cells in Rgr-d mice showed apical displacement of intracellular pigment and appeared vacuolated compared to RPE in the wildtype littermate. Similar changes were not seen in RGR knockout mice.
In contrast to the RGR-d isoform in humans, mouse RGR-d undergoes rapid degradation, and abnormal RGR-d protein does not accumulate in the eyes of otherwise healthy, untreated young mice. These animals provide a model for lifelong expression of an Rgr-d gene and potentially chronic endoplasmic-reticulum stress or age-related proteopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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