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C.M. Donmoyer, V.T. Ciavatta, G.I. Liou, J.M. Nickerson; Microarray Analysis Of The Neonatal Irbp Knockout Mouse . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4895.
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© ARVO (1962-2015); The Authors (2016-present)
The juvenile IRBP knockout (KO) mouse has a ∼50% reduction in ERG a–wave amplitude and experiences a slow retinal degeneration that continues throughout its lifetime. We sought to find early changes in the expression of genes, as changes among these genes may reflect regulation of networks in which IRBP participates. These genes may be associated with normal IRBP function or may compensate for the loss of IRBP expression.
Retinas were dissected to exclude lens, cornea, iris, RPE, choroid, and sclera from p7 and p10 IRBP KO and C57BL/6 (WT) mice. About 20 retinas per group were extracted in RNAsol. RNA intactness was assessed by capillary electrophoresis at Expression Analysis, where Affymetrix 430a GeneChips were prepared. Functional groupings of altered genes were assessed using the program, DAVID, v2.1.
RNA was intact and at the expected concentration, and experiments were performed in duplicate. As controls, IRBP mRNA was at least 4 fold reduced in KO groups, consistent with expected values. Expression levels of GDF2, GDF10, and Annexin 8, the genes immediately upstream and downstream of IRBP, were unaffected by the interruption of IRBP. At p7, about 1000 genes were expressed at significantly (p<0.05) different levels in the KO compared to WT, while at p10 about 4000 genes were changed significantly. According to the program DAVID, among the 146 genes that deviated 2–fold from WT expression levels at p7, 3 families of genes were upregulated and 4 families were downregulated. At p10, about 20 families of genes were upregulated and 20 were downregulated, among the 1086 genes that deviated 2 fold from WT. Expression of rds–peripherin was reduced 20–fold, while rom–1 was unaltered, suggesting that rds:rom–1 heterotetramers may require IRBP for assembly. Crystallin genes were profoundly downregulated in the p7 KO mouse, suggesting a role in the development and assembly of outer segment disks, which appear "ragged" in the KO. Several kinase–mediated cascades are altered when IRBP expression is missing, as several constituents are up– or downregulated, which suggest that IRBP may be involved in feedback loops.
The much smaller number of differently expressed genes in the KO mouse at p7 than p10 suggests that p7 is near the optimum age to identify the earliest altered genes in the absence of IRBP, consistent with the lack of noticeable morphological changes at these ages. The small number of affected functional groupings suggests that IRBP is required in several pathways to permit proper development of the retina.
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