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C. Lupien, C. Salesse; Identification of Genes Specifically Expressed in Human Muller Cells by Subtractive Hybridization . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2260.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Müller cells are the predominant type of glial cells in the retina. These cells play very important functions in the retina. The purpose of this study was to generate a profile of the genes expressed in the human retinal Müller cells and identify genes that may be responsible for retinal diseases. Methods: Subtractive hybridization is a technique that enables to compare two populations of mRNA and obtain clones of genes that are expressed in one population but not in the other one. A cDNA subtraction library was constructed using RNA isolated from human primary cultures of Müller cells and human astrocytes. The PCR-select cDNA subtraction kit (Clontech) allows to selectively amplify differentially expressed genes. The cDNA of Müller cells is called "tester cDNA" and the reference cDNA from the astrocytes is called "driver cDNA". Tester and driver cDNAs are hybridized and the hybrid sequences are removed. Consequently the remaining unhybridized cDNAs represent genes that are expressed in the tester, but are absent from the driver mRNA. Dot blot arrays of clones from the subtracted library are hybridized with labeled probes from either tester or driver populations. Clones that are recognized by the tester probe and not by the driver probe are confirmed to be differentially expressed. Results: We obtained 1342 clones, 777 of them were hybridized with 4 different probes (tester and driver probes, cDNA of Müller cells and cDNA of astrocytes). We identified 246 genes specifically expressed in human retinal Müller cells, 141 of them had previously been characterized, the others ones correspond to genomic clones or hypothetical proteins. Some of these are known as retinal expressed genes and include genes involved in cellular metabolism, cell proliferation, cell adhesion, cytoskeleton, extracellular matrix, growth factors, transport proteins, transcription and translation proteins. Several of these genes map to the chromosomal regions of previously localized retinal disease loci and could be suspected as candidate disease genes. Conclusions: The analysis of the subtraction library revealed genes that are specifically expressed in human retinal Müller cells. Some of these genes are unidentified, novel genes that are specific to Müller cells which thus represent candidate genes for retinal diseases.
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