The methylation-free state of the paternal ICE depends on the CGI.

<p>(A) DNA blot assaying methylation of the <i>Airn</i> promoter MluI site as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a>, in undifferentiated ES cells carrying a paternal <i>CGIΔ</i> or wildtype (<i>+</i>) allele. The 5.0 kb band identified by probe MEi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map) indicates a gain of methylation on the <i>CGIΔ</i> paternal allele. This band is weaker in cells with lower passage numbers that still retain the selection cassette (<i>S12/CGIΔ+cas</i>-1,-2) compared to cells that have been in culture for 8 more passages (<i>S12/CGIΔ</i>-1A,-1B,-2A,-2B) with a deleted selection cassette. The lower panel confirms this by showing a matching loss of the unmethylated 1.1 kb fragment specific to the paternal allele in cells with a higher passage number. Both panels were from the same blot and the intervening area lacking any hybridisation signal removed. (B) DNA blot as in (A) assaying <i>Airn</i> promoter MluI methylation during ES cell differentiation showing that the level of paternal methylation on the <i>CGIΔ</i> allele in undifferentiated ES (d0) cells (5 kb band) does not change in differentiated d5 and d14 cells. Probe MEi is a 1 kb EcoRI-MluI fragment shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map. (C) Bisulfite sequencing of two undifferentiated <i>S12/CGIΔ</i> ES cell clones using primers spanning the deletion that specifically amplify the paternal <i>CGIΔ</i> allele, confirms the strong gain of DNA methylation, but also shows that some alleles are more methylated than others (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5B</a>).