Figure 3. The change of genomic imprinting during VSEL ageing. Panel A. RQ-PCR analysis of RasGrf1, Igf2, and H19 expression in VSELs isolated from 4-week-old and 18-month-old C57BL/6 mice. The relative expression level was represented as the percentage of β2 microglobin (β2mg) expression and shown as the mean ± SD. Please note reciprocal expression in Igf-2 (that promotes proliferation) and H19 (that encodes for mRNA giving rise to several miRNA negatively affecting cell proliferation). Both of these genes (Igf2-H19) are regulated in tandem by a common DMR that affects their expression - when one is silenced another one is upregulated. Panel B. Combined bisulfate-restriction analysis (COBRA) assay of Igf2-H19 DMR1 (upper panel) and RasGrf1 DMR (lower panel) by BstUI restriction enzyme in 6-month-old control wild type (WT) and 6-month-old transgenic bovine GH-overexpressing mice (bGH-TG). The COBRA assay was performed on VSELs, hematopoietic stem cells (HSC), and bone marrow mononuclear cells (BMMNC). “-” indicates bisulfate modification was not performed on the DNA (control); “+” indicates that DNA was subjected to bisulfate modification. The unmethylated DNA (uMe) was not cleaved, in contrast to methylated DNA (Me), because of a sequence change at the site recognized by a restriction enzyme after bisulfite reaction.