The Analysis of Differentially Expressed Gene by DNA Micro Array

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We have conducted histology, molecular biology, and genetic analysis of macular degeneration cynomolgus monkey with drusen. This monkey family previously discovered in National Primate Center in Tsukuba has been briefly characterized by observation of the retina and fluorescence imaging of the retina section. Sixty monkeys aged 15 to 24 years in propagation colonies were examined by fundus scope. Macular degeneration was observed in ten monkeys. Histological sections prepared from excised macular retina were stained by HE (hematoxylin and eosin), PAS (Periodic Acid Sciff), LFB (luxol fast blue) and MT (masson trichrome) and observed under light microscopy. Ultrastructure of drusen was examined by transmission electron microscopy. Hyper-pigmentation, partial vacuolation, increased autofluorescence, and PAS-positive granules in cell cytosol were observed in retinal pigment epithelium (RPE) of these affected monkey retina. However, minimum histological abnormality was observed in neural r … More etina and choroid. These observations indicate that lipofuscin granules are highly accumulated in RPE.. Molecular biology analysis using Clontech Inc, plastic DNA array with 8,000 genes were performed by labeling RNA extracted from affected and non-affected monkey retina by Cy3 and Cy5 respectively. After the normalization of all 8,000 genes, two samples were compared for differentially expressed gene. Fourteen genes were up-regulated by, 2.5 to 7.6-fold, while, five genes were down-regulated by 2.4 to 3.8-fold. Among the up-regulated genes, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2 gave more than 7.6-fold compare to normal. Lymphatic vessel endothelial hyaluronan receptor gave significant drop of 3.8-fold. Linkage analysis and candidate gene approach was performed in parallel to determine the gene responsible for the disease. The monkey homologue of the ELOVL4, gene responsible for autosomal dominant Stargardt-like macular dystrophy (STGD3) was cloned to determine any possible mutation in macular degeneration monkey. PCR of genomic DNA and 5'/3'-rapid amplification of cDNA ends were carried out to determine the complete sequence of the monkey ELOVL4 cDNA and the gene. ELOVL4 RNA was abundantly expressed in retina and thymus determined by real time quantitative PCR. Immunohistochemical analysis was performed using anti-human ELOVL4 peptide polyclonal antibody to localize ELOVL4 protein in monkey retina. No mutation was found in this gene. Less