Fig 4. Effects on the DNA methylation and imprinting status of the Peg3 domain.

(A) Schematic representation of the Peg3 domain showing the relative positions of 4 genomic intervals that have been analyzed for their DNA methylation status. The relative position of the region targeting the Peg3-DMR is illustrated in detail within a box. The 400-bp region surrounding the 1st exon of Peg3 was analyzed, which is localized within the inverted region of the KO allele. (B) A series of DNA methylation analyses were performed using the DNA isolated from the neonatal heads. The isolated DNA was treated with the bisulfite conversion protocol, and subsequently used for the amplification of each target region. The amplified PCR product was analyzed with COBRA. The restriction enzyme used for each set of PCR products is shown underneath of the name of each target. The unmethylation and methylation based on the digestion pattern by a given restriction enzyme are also indicated by the letter U and M with arrows, respectively. (C) RT-PCR-based imprinting test of the genes within the Peg3 domain. This series of imprinting tests used the total RNA isolated from the neonatal heads of the F1 hybrid of the male set that had been prepared through the reciprocal crossing of the heterozygotes with the 129/B6 background and the breeding partners with the PWD/PhJ background. The products from RT-PCR were digested with a given restriction enzyme to differentiate parental alleles, which are shown as different-size DNA fragments on gel images. The two columns on the left represent the digestion patterns for two parental strains for each gene; the two middle columns represent the results from the F1 hydrid set with the paternal transmission of the KO allele (male heterozygote with female PWD/PhJ); the two columns on the right represent the results from the F1 hybrid set with the maternal transmission of the KO allele (male PWD/PhJ with female heterozygote).