Figure 1.

The overview of the Peg3 domain and targeting scheme. (A) Schematic representations of the Peg3 domain (upper panel). Each imprinted gene is indicated with an arrow. The genes with pink are paternally expressed genes, whereas the genes with blue are maternally expressed genes. The three DMRs are indicated with gray boxes. Targeting scheme (lower panel). The 4.0 kb Peg3-DMR contains the first exons of Peg3 and Usp29 and two evolutionarily conserved elements (CSE1 and CSE2 or YY1-binding site) that are located within the first intron of Peg3. The current targeting scheme was designed to delete the 2.5 kb genomic region harboring CSE1 and YY1-binding sites. The transcriptional direction of Peg3 and Usp29 is indicated with arrows, and exons are indicated with thick vertical lines. The region corresponding to the neomycin resistance gene (Neo) along with the two flanking loxP sites within the targeting vector are also indicated by an open box and triangles, respectively. Arrows with numbers underneath ‘Targeted allele’ indicate primers with relative positions that were used for long-distance PCR. (B) Southern blot analyses using the DNA isolated from ES cells that have been transfected with the targeting vector. BamHI-digested DNA was hybridized with the 5′-side probe, showing an additional 5 kb fragment in a targeted clone (marked with *). (C) Western blot analyses using the protein extracts prepared from the neonatal brains of WT and KO pups. This analysis revealed at least 3–4-fold down-regulation of Peg3 in the pup inheriting the KO allele paternally. (D) Picture of 5-day-old WT and KO pups of paternal transmission.