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M. Alizadeh, W. Feng, D. Vollrath; Global Search for Genes Involved in Phagocytosis of Photoreceptor Outer Segments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2290.
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Purpose: Outer segments grow and are shed in a circadian fashion. In light-entrained rats and mice, a burst of rod disc shedding and RPE phagocytosis occurs soon after lights on. We investigated global gene expression patterns in the retinas of mice at time points during the day that correspond to different levels of disc shedding and RPE phagocytosis of OS membranes. We also examined gene expression patterns during RPE phagocytosis of OS in cell culture. Methods: Mice were raised under 12-hr light-dark cycle until approximately 2 months of age. The initial time points examined were the approximate peak (ZT 1.5), and the approximate nadir (ZT 16) of phagocytosis. Eyes were removed immediately after sacrificing the animals. The removal and dissection of eyes at night were conducted in dim red light to prevent possible changes in gene expression due to white light. Neural retina was dissected from RPE/choroid/sclera and total RNA was extracted from each sample. Two-color competitive microarray hybridizations were performed on glass slides spotted with a set of 40,000 mouse cDNA clones comprising 25,000-30,000 unique genes. For cell culture studies, RPE cells were incubated with purified OS for various lengths of time, and total RNA was isolated and converted to labeled cDNA, followed by standard microarray hybridizations. Results: Preliminary data from three separate experiments (ZT 1.5 vs. ZT 16) indicate that, while most genes show no significant change, a small number of genes are induced or repressed by at least 2-fold in RPE/choroid/sclera. Data from RPE phagocytosis of OS in cell culture identify genes with similar expression patterns in vivo. Conclusions: The use of microarray technology is likely to identify important genes involved in RPE phagocytosis of OS. Genes that have similar expression patterns both in vivo and in cell culture could be involved in RPE phagocytosis and will be chosen for further studies. This approach will extent our understanding of the molecular mechanisms of OS disc shedding and RPE phagocytosis. Abbreviation: Zeitgeber Time (ZT)
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