MOESM3 of Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus

Additional file 3: Figure S3. Introduction of 116-bp deletional mutations within the transgenic or endogenous H19 ICR in mice by CRISPR/Cas9 genome editing. (A) Sequence alignment of wild-type and the mutant H19 ICRs. Protospacer-adjacent motif (PAM) and gRNA sequences are shaded and underlined, respectively. Cleavage sites predicted by PAM locations (arrowheads), as well as the end positions of del-5-9 fragments are shown. (B) Partial restriction enzyme maps of the endogenous H19 locus and the β-globin YAC transgene carrying the H19 ICR fragment with the 116-bp deletion. Methylation-sensitive HhaI sites in the BamHI (B) fragments are displayed as vertical lines beneath each map. The probe used for Southern blot analysis in (C) is shown as a filled rectangle. B; BamHI, G; BglII, H; HindIII, Sa; SacI sites. (C) DNA methylation status of the mutant H19 ICR fragment in somatic cells of the YAC-TgM that inherited the transgene either paternally (pat.) or maternally (mat.). Tail DNA was digested with BamHI and then HhaI, and the blot was hybridized with the probe shown in B. endo.; endogenous locus, Tg; transgene. Asterisks indicate the positions of parental or methylated, undigested fragments.