Gene Expression Induced By Hyperoxia In The C57bl/6j Mouse Retina

Abstract

Abstract: : Purpose:To identify changes in gene expression induced by the exposure of the retina to sustained hyperoxia, in the C57BL/6J mouse. Methods:2 groups of 6 mice were placed in constant 75% oxygen for a period of either 1 or 2 weeks. A control group also containing 6 mice were exposed to normal oxygen conditions. Total RNA was extracted from the retinas, purified and 8µg of purified total RNA were used in the Affymetrix GeneChip® protocol (www.affymetrix.com) to create biotin–labelled cRNA. The labelled cRNA created for each time point was then hybridised to an individual Affymetrix GeneChip® U72v2A (approximately 6000 characterised genes and 6000 EST clusters). Patterns of gene expression at the 3 time points were then compared using the Affymetrix MAS 5 software. Genes were considered significantly regulated when the fold change between time points equalled or exceeded 4. Results:Comparing gene expression at 1w to controls, genes significantly down regulated outnumbered genes significantly up regulated by 111 to 25. Comparing 2w to controls, a different pattern emerged; genes down regulated were much less numerous than those upregulated (8 to 71). Comparing gene expression at 1w vs 2w, expression was up regulated in 63 genes, and down regulated in 8 genes. Among the genes significantly up regulated at both 1w and 2w were glial fibrillary acidic protein (GFAP) and cytoplasmic ß–actin. Several members of the crystallin family of heat shock proteins were also strongly regulated at both 1w and 2w. One, mu–crystallin, was up regulated, while crystallin α–A, crystallin ß A3, crystallin γ B, C, d, E, F and S were all down regulated. 5 of the 8 genes that were progressively down regulated from 1w to 2w were crystallins. Conclusions:The Genechip®, and the significance criterion (4–fold change) used, identified approximately 100 genes up or down regulated by sustained hyperoxia. Data indicate that during the 2w of sustained hyperoxia, the regulation of significant numbers of genes changed; i.e. their up–or down regulation was transient. This analysis provides a platform for more specific analysis of genes regulated by hyperoxia. Because sustained hyperoxia is a feature of the end–stages of many, perhaps all photoreceptor degenerations, this analysis may give insight into the mechanisms that make many degenerations progressive and non–specific.

Keywords: gene microarray • retina • crystallins