Identification of Genes Expressed in Cone Photoreceptor Cells

Abstract

Abstract: : Purpose: To identify genes expressed exclusively or preferentially in cone photoreceptors that may be candidates for the cause of retinal degeneration in animals and humans. Methods: The retina of adult cd dogs is totally devoid of cone photoreceptors due to inherited retinal disease. We took advantage of this retina and subtracted its mRNAs from those of the normal dog retina using two rounds of a PCR–based method (RDA). The output of RDA was then shotgun cloned into a plasmid vector to create a mini–library in a bacterial host. Approximately 2,000 cDNA clones generated from the subtracted library were arrayed on microarray chips after amplification of inserts from individual colonies with vector–specific primers. The arrayed target cDNAs were hybridized with the fluorescent–labeled original amplicons from normal and adult cd dog retinas. The array was then screened by repetitive probing with mixtures of the inserts from the arrayed library that had the brightest signal after hybridization with the initial amplicons, to create a non–redundant set of clones. Results: : 80 differentially expressed, non–redundant clones were sequenced. BLAST analyses were performed utilizing the National Center for Biotechnology Database (www.ncbi.nlm.nih.gov/BLAST) and the Institute for Genomic Research (www.tigr.com) for homology searches and SAGE Genie (http://cgap.nci.nih.gov/SAGE) for sage tag research in peripheral versus macular expression in human retina. Out of these 80 sequenced clones, we identified several that have been described as cone specific, and many that did not correspond to any known characterized gene or to genes that have not been described as present in the retina. Northern blots and real–time RT–PCR have confirmed the differential expression of the isolated cDNAs. Conclusions: The combination of PCR–based cDNA subtraction with cDNA microarrays has provided us with an efficient way to identify genes expressed in cone photoreceptors. Characterization of these clones is in progress.

Keywords: retina • gene microarray • genetics