Fig. 8

Genomic imprinting recapitulated in LCb118 knock-in mice. a Breeding scheme. In order to distinguish parental origin of the alleles by using SNPs between inbred mouse strains, endo-LCb118 homo-knock-in mice (LCb118/LCb118; C57BL/6 J [B6] background) were mated with wild-type mice (H19 ICR/H19 ICR; JF1/Msf [JF1]), and offspring was obtained. b–d Livers from two pairs of E18.5 embryos, each inheriting the knock-in allele either paternally (pat.; P) or maternally (mat.; M) were used for genomic DNA, total RNA, and chromatin preparations, as in Fig. 6. b DNA methylation status of LCb118 region (the same position as in Fig. 7a) was analyzed by bisulfite sequencing. c ChIP analysis of CTCF occupancy at the LCb118 sequence. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following qPCR analyses of three distinct genomic regions (Necdin; negative control, endogenous H19 ICR; positive control, and LCb118), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted. Statistical differences were determined using an unpaired t test (N.S., not significant). d The allele-specific expression of the Igf2 and H19 genes was examined by RFLP analysis. Igf2 and H19 gene transcripts were amplified by RT-PCR followed by BstUI or Cac8I digestions, respectively. Parental origin of transcripts was discriminated by allele-specific restriction sites. The sites were also introduced into primer sequence so that complete digestion of PCR products can be concomitantly monitored. e Schematic representation of the genomic imprinting recapitulated in the LCb118 knock-in allele. f Hypothetical model for post-fertilization methylation maintenance mechanism at the Igf2/H19 locus