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Cynthia X. Wang, Kaiyan Zhang, Bogale Aredo, Rafael Ufret-Vincenty; Differential gene expression of RPE cells in Cfh transgenic mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6503.
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The 402H variant of complement factor H (Cfh; a key regulator of complement activation) increases the risk of AMD up to 7-fold. Our goal in this work is to determine how changes in Cfh affect the expression of RNA by RPE cells as mice age.
We generated a transgene consisting of human Cfh SCR6-8 flanked by mouse Cfh SCR1-5 and SCR9-20 (under the ApoE promoter). The resulting chimeric CfhTg mice were crossed to mCfhKO mice (deficient on mouse Cfh). We developed a new method for isolation of RPE cells from eyecups, and used it to isolate RNA from RPE cells of CfhTg/mCfhKO vs B6 mice. We ran an Illumina microarray chip on these samples, and performed a pathway analysis on the resulting genes. We chose a subgroup of these genes for RT-PCR confirmation. We also chose an independent group of candidate genes for RT-PCR analysis.
RPE cells were isolated from 1 year old CfhTg/mCfhKO mice and age-matched B6 mice using a new simple method. RNA was then extracted using the Qiagen RNeasy micro kit. Bioanalyzer testing confirmed the high quality of the RNA. RT-PCR for RPE65 and PECAM confirmed the purity of the RPE cell population. Analysis using an Illumina microarray chip generated a list of 96 genes that were differentially expressed by 2-fold or more in CfhTg/mCfhKO mice. Pathway analysis revealed that among the top canonical pathways identified were those involving leukocyte extravasation signaling and chemokine signaling. Pathway data and RT-PCR confirmation will be presented.
Variants of Cfh lead to changes on the gene expression profile of RPE cells. Future work will focus on determining whether these changes in gene expression by RPE cells promote disease in the retina/RPE.
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