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N Kociok, U Schraermeyer, TT Luther, G Thumann, JF Jordan, P Esser, B Kirchhof; Identification of Differentially Expressed Genes in RPE Cells of Dystrophic RCS Rats With Possible Connection to Age Related Macular Degeneration in Man . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2806.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: For individuals over 50 years AMD, age-related macular degeneration, is the leading cause of central vision loss in western nations. AMD is a disease with a multi factorial inheritance in which environmental factors can trigger disease in those who are 'genetically primed'. An exact animal model for AMD does not exist. In the dystrophic Royal College of Surgeons (RCS) rat retinal degeneration is caused by a mutation in the gene for c-mer tyrosine kinase. This impairs RPE phagocytosis of retinal outer segments leading to progressive loss of photoreceptors. We used RCS rats to search for genes in RPE cells with changed expression due to impaired phagocytosis. Homologous human genes may have relevance for degenerating diseases of the human eye. Methods: Pure RPE cell sheets of dystrophic and nondystrophic RCS rats at day18 were prepared using Ca2+-free/EDTA solution and a differential mRNA expression analysis was performed. Differentially amplified amplicons were cut out of the gel, the cDNA cloned, sequenced and identified by gene data bank homology searching. The differential expression of selected genes were verified by semi quantitative reverse transcription-PCR. Results: An isolation method for pure RPE cells sheets without contamination by other cells was established. Differential mRNA expression analysis on total RNA with a subset of all possible combinations of 12 differential display primers revealed a different expression of 11 cDNAs in dystrophic RCS rats when compared to nondystrophic controls. Six amplicons showed a homology to mouse genes, the corresponding rat genes are not yet in the gene data bank. Five amplicons were identified as rat genes with limited functional characterization. The expression of these five known rat genes (SID1669P, CRM1/exportin 1, CDK109, INSIG1/CL-6, MYAK) in dystrophic and non-dystrophic RCS rats were confirmed and quantified by semi-quantitative RT-PCR. Conclusion: From the limited information on the function of the detected genes one can conclude that they are involved in cellular functions like nuclear export of proteins and mRNAs (SID1669P and CRM1/exportin 1), modulation of growth and differentiation of tissues involved in metabolic control (INSIG1/CL-6), and rapid degradation of proteins (MYAK). Mutation analysis of the human homologues of these genes will reveal whether they can be correlated with the onset or severity of AMD, eventually helping to design early treatments to delay the onset of symptoms or alleviate the phenotype of retinal degeneration diseases.
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