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Jutta U. Hosch, Stefan Günther, Thomas Braun, Alfred M. Pingoud, Eveline Baumgart-Vogt, Birgit Lorenz, Knut Stieger; Development of a Humanized Mouse Model for X-linked Retinitis Pigmentosa caused by a Point Mutation in the Rpgr Gene. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2360.
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Mutations in the gene encoding the retinitis pigmentosa GTPase regulator (RPGR) are the most frequent cause for X-linked RP (XLRP) in man. Most mutations can be found in a specific repetitive region of the retina-specific terminal exon open reading frame (ORF) 15. Point mutations in the ORF15 cause a frame shift, leading to a modified C-terminal amino acid chain and consequently a toxic gain of function of the altered protein. The purpose of this study was to develop a mouse model that contains a 1-bp deletion at position 2793 in RPGR-ORF15, representing a typical human mutation and inducing a change of the amino acids at the C-terminal end from the EG rich motif to the RK rich motif.
The pathologic mutation and several silent point mutations were introduced into murine embryonic stem (ES) cells by homologous recombination using a targeting vector. In addition, an ISceI homing-endonuclease recognition site was introduced. Modified ES cells were injected into BL6 blastocytes and implanted into surrogate mother mice (Balb/c). F1 offspring was crossbred with Cre-deleter mice (BL6) and subsequently crossed into BL6 background. RNA expression analysis of the mutated allele, histology and immunohistochemistry were performed for initial phenotypic analysis at different time points after birth.
Two transgenic mouse lines (derived from clones 160 and 320) were established, which propagate the desired mutation within the ORF15, causing a shift from the GE rich motif to the RK rich motif at the protein level. The level of the RPGR-ORF15 splice variant mRNA in the retina remained unchanged. Histological analysis of the retina at 3 months showed a clear reduction in thickness of the photoreceptor and inner nuclear layers in affected animals compared to wild type control mice. Delocalized pycnotic nuclei within the inner and outer segments of the photoreceptors were observed.
At 3 months, this humanized mouse model of XLRP shows definitive, yet moderate signs of retinal degeneration similar to the situation in humans. Therefore, it will be useful to gain further insight into the pathological mechanisms involved in the degenerative process and the biochemical reason for the toxicity of the altered protein. In addition, it will be possible to use the model for developing new therapeutic strategies for the treatment of XLRP in humans.
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