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Leonard M. Hjelmeland, Alfred K. Yu, Zeljka Smit-McBride; Age-related Variation of Cytosine Methylation in the Sod2 Promoter in the RPE/choroid and Retina Within and Among Individual BALB/c Mouse Eyes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5136.
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© ARVO (1962-2015); The Authors (2016-present)
To quantify the variation in the methylation of cytosines within a single individual and among a cohort of isogenic members of the BALB/c strain as a function of age. For this purpose, high resolution bisulfite sequencing of the Sod2 promoter at the 20X level was performed on gDNA samples prepared from the RPE/choroid and retina of the BALB/c mouse eye.
BALB/c mice were purchased from Jackson Laboratories (2 months) or the NIA aging animal colony (18 months). Animals were housed and fed according to the ARVO guidelines. Genomic DNA was prepared from freshly dissected RPE/choroid and retina of 3 replicate animals 2 or 18 months of age from both males and females. These samples were treated with sodium bisulfite. Selected regions of the Sod2 gene (promoter and intron 2) were amplified by PCR and cloned. At least 15-20 individual clones for each amplified region were sequenced and these data were used to calculate percent methylation at each cytosine residue. The mean values for each position were then averaged among 3 different animals. Data were tested for significant differences among groups.
Variance in the methylation of individual cytosines was itself also variable with respect to each individual residue examined in the Sod2 sequence. When data were averaged over 3 biological replicates the same trends in variance were observed. No individual cytosine showed significant differences among groups at the p< 0.05 level, but several residues near to NFkappaB and FOXO3A binding sites in the proximal promoter as well as a region in the 2nd intron trended towards significance (i.e. p< 0.07 to p<0.09).
Our results suggest that the experiments were underpowered and that increasing the number of biological replicates and/or depth of sequencing may yield statistically significant results. Significant alterations in the cytosine methylation near transcription factor binding sites could affect gene expression in an age-related manner.
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