Abstract
Purpose:
Recent studies have indicated that SNCG is a marker for retinal ganglion cells (RGC) and also that loss of RGC correlates with loss of Sncg in various mouse models of glaucoma. In this study we used novel systems genetic methods, mathematical modeling, flow cytometry and siRNA transfection techniques to identify and evaluate the candidate gene regulating Sncg in primary murine RGCs.
Methods:
Quantitative trait locus (QTL) mapping was used to identify the genomic region that regulates Sncg expression. Correlation analyses (direct and partial Pearson test) were performed and SNPs were evaluated to identify the candidate gene(s) that regulate Sncg expression. We determined SNCG and PFDN2 cellular localization by immunohistochemistry from mouse retina tissue. Additional functional assays were performed from cell sorted live primary RGCs isolated from murine retina by flow cytometry and cultured ex vivo. To better understand RGC proliferative capacity we employed a carboxyfluorescein succinimidyl ester (CFSE) based cell proliferation assay where cell division is measured by CFSE intensity. siRNA transfection was performed using siRNA specific for Pfdn2, followed by Western blot analysis and qPCR to validate the effect of knockdown of Pfdn2 on Sncg expression in RGCs.
Results:
Simple interval mapping showed a significant trans-eQTL for Sncg on chromosome 1 in the QTL-rich region (Qrr1) with a peak likelihood ratio statistic (LRS) of 34.1. Partial correlation analysis while controlling for the Qrr1 revealed a single candidate, Pfdn2, a gene involved in protein folding. Pfdn2 correlation with Sncg is significant (r=0.609, p=3.38x10-9). Our SNP data revealed one non-synonymous SNP on exon 1 and six indels in Pfdn2 between the parental mice strains C57BL/6J and DBA/2J. Immunostaining studies reveal SNCG and PFDN2 are co-expressed, supporting our hypothesis that Pfdn2 regulates Sncg. Cell proliferation studies revealed 8.8% of total sorted RGC proliferate by 96-hrs of culture. We are optimizing our studies and testing the effect of Pfdn2 inhibition by Sncg siRNA transfection through qPCR for gene expression and Western blot for protein analysis.
Conclusions:
Our study highlights mouse systems genetics analysis approach, identifies and verifies Pfdn2 as a gene regulating Sncg expression in primary mouse RGCs.
Keywords: 529 flow cytometry •
531 ganglion cells •
534 gene mapping