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Cynthia Wang, Bogale Aredo, xiao chen, Rafael Ufret-Vincenty; Differential gene expression of RPE and choroid in old Cfh transgenic mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6192.
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Our goal in this work is to determine how variants in complement factor H (Cfh) and the levels of c-reactive protein (Crp) affect the expression of RNA by RPE cells and/or their choroid as mice age.
A transgenic mouse was generated which expresses chimeric Cfh molecules consisting of human Cfh SCR6-8 flanked by mouse Cfh SCR1-5 and SCR9-20 (under the ApoE promoter). These mice were crossed to mCfhKO mice to eliminate mouse Cfh. We also generated some lines by crossing the resulting CfhTg/mCfhKO mice to hCrpTg mice, which express human Crp. Usinga method we developed, we isolated RPE cells from mouse eyecups and isolated RNA from RPE cells of 2 year old CfhTg/mCfhKOvsCfhTgCrpTg/mCfhKO mice vs B6 mice. In addition, we isolated RNA from the choroid/sclera. We ran Illumina microarray chips on these samples, and performed a pathway analysis on the resulting genes. We re-tested these genes using RT-PCR. We also chose an independent group of candidate genes for RT-PCR analysis.
RPE cells were isolated from 2 year old CfhTg/mCfhKO mice, CfhTg/hCrpTg/mCfhKO mice and age-matched B6 mice using a new simple method. RNA was then extracted from the RPE cells using the QiagenRNeasy micro kit. RNA was also extracted (separately) from the residual choroid/scleral cup. Bioanalyzer testing confirmed the high quality of the RNA. Analysis using Illumina microarray chipsgenerated a list of genes that were differentially expressed in CfhTg/mCfhKO mice or CfhTg/hCrpTg/mCfhKO when compared to B6, either in the RPE cells or in the choroid. RT/PCR analysis was used to confirm the results and look at additional candidate genes.
Variants of Cfh, and the expression of Crp, both lead to changes on the gene expression profile of RPE cells and choroid in aging mice. Protein expression assays will be done in the future to confirm these gene expression changes.
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