(a,b) COBRA analysis showing extensive demethylation of H19/Igf2 ICR and KvDMR1 (an intronic CpG island within the KCNQ1 gene) in P7-ESCs. Bisulfite converted DNA was amplified with bisulfite specific primers and methylation content was assessed by digestion with BstUI that recognizes CGCG sequences. Digestion of BstUI serves as indicator of the presence of methylated sequences which were resistant to C to U conversion. Bisulfite sequencing of KvDMR1 (b) supports the lack of DNA methylation measured by COBRA assay. (c) Example of changes in transcription of two imprinting regulated genes in P7-ESCs. Experiments in a and b were repeated independently twice.