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SE Oviatt, C Naber, R Grewal, DT Organisciak, R Darrow, L Barsalou, P Wong; Generating the Molecular Tools to Define Gene Expression Patterns Associated With Increased Susceptibility of the Retina to Light Induced Degeneration . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1445.
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Purpose: It has previously been shown that there is a circadian component to retinal light damage susceptibility. Rats exposed to high intensity green light starting at 1AM show extensive photoreceptor loss, while rats exposed to light starting at 5PM show little photoreceptor loss (Organisciak et al. IOVS 2000 41:3694-3701). We have compiled an EST collection representing genes that are expressed in the rat retina at 1AM. Our goal is to identify genes that may play a role in the increased susceptibility of the retina to light induced damage. Methods: A 1AM dark-reared rat retina cDNA library was made in the Lambda Zap II vector system. Clones were isolated at random to generate a clone bank. The presence of a cDNA insert in excess of 600 bp in each isolated clone was verified by PCR and electrophoretic analysis before the clone was included in the clone bank. PCR amplified inserts from each clone were physically arrayed on to a nylon membrane and differentially cross-screened with cDNA probes representing the 1AM and the 5PM dark reared retina. In addition, a number of the banked clones were randomly selected for single-pass sequencing and were analyzed using the Blast program. Results: Approximately 800 clones were isolated and banked. Based on the array analysis, 15% of the genes screened appear to be differentially expressed at 1AM and 5PM. Included in the clones that have been sequenced were clones known to be involved in normal retinal function, as well as rat orthologues of mouse and human genes. Approximately 10% of the clones analysed for DNA sequence represent genes with unknown function. Conclusion: We have isolated clones from a 1AM rat retinal library and have identified genes that are putatively differentially expressed at 1AM. Northern analysis will be done to confirm this differential expression. Additional work is needed to determine if the expression of these genes plays a role in the increased susceptibility of the retina to light induced photoreceptor cell degeneration.
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