Fig. 4. (ChßGI)2 methylation. (A) Southern blots of tissues of 18.5 dpc fetuses. L, liver; M, carcass (mostly muscle); K, kidney. This assay tests for methylation at the central methylation-sensitive Hpa (HpaII) (CCGG) and Hha (HhaI) (GCGC) sites indicated below the blots (if methylated, the 2.6 kb Eco (EcoRI) and Bam (BamHI) band should remain intact). Msp (MspI) cuts at CCGG, regardless of CpG methylation. The CTCF sites are indicated (vertical arrows). The 2.6 kb Eco, Bam fragment consists of the 2.4 kb (ChßGI)2 plus an additional 0.2 kb of downstream sequence (see Fig. 2). Major bands expected on complete cutting are shown below blots. The complete ChßGI sequence was the probe used. Number of embryos used: +/–(P), one embryo per lane for L and M, and three embryos per lane for K; –(M)/+, one embryo per lane for L and M, and two embryos per lane for K. (B) Purification and methylation state of germ cells of 18.5 dpc +/–(P) fetuses. In the flow cytometry graph, EGFP+ cells above the line were sorted for analysis. Each row of squares represents the methylation state of each of 22 sequential CpGs, 5'
3' orientation as in Fig. 1B (white square, unmethylated; black square, methylated). Each row represents a separate sequencing reaction. The CpG of the CTCF site is indicated (arrow).
