Figure 3.

Schematic representation of the 5'-flanking region of the murine glut1 gene is shown above with locations of the proximal CpG island and the primers (p) employed in methylation specific PCR with the amplification product size in parenthesis. Representative gels demonstrating the amplification products of the glut1 5'-flanking region in control (CON; top panel) and calorie restricted (CR; bottom panel) groups by methylation specific PCR (MSP) are shown (n=5 placentas from different pregnant mice). Amplified products recognized by methylated (M) and unmethylated (U) primers are seen. Relative frequency of methylation and unmethylation in CON (bottom left panel) and CR (bottom middle panel) groups was calculated based on the optical density of the amplification products (arbitrary units) as a ratio of M+U value for a given sample (i.e. M=M/M=U or U=U/M+U) represented as a percent. n=5/group, *p value as shown in each graph when compared to the corresponding CON group. The bottom right panel shows a graph that depicts the relative frequency of methylation (M) in CON and CR groups with the methylation frequency compared between the CON and CR groups, n=5/group. This was followed by bisulfite conversion and sequencing which demonstrated no methylated CpG sites located 5'- to the ATG region in response to CR (negative data not shown).