Fig. 1

Generation of rat H19 ICR and mouse IG-DMR transgenic loci at the identical chromosomal site. A Structure of the rat Igf2-H19 gene locus. Monoallelic expression of paternal Igf2 and maternal H19 genes depends on the shared 3' enhancer and methylation state of the H19 ICR, that is methylated (solid circle) and unmethylated (open circle) at the paternal and maternal alleles, respectively. In the enlarged rat H19 ICR map (rH19 ICR), CTCF and Sox/Oct binding sites are indicated by dots (1–4) and a solid box, respectively. The 113-bp sequence homologous to the “mouse 118-bp sequence” is denoted as gray box. B Structure of the mouse Dlk1-Dio3 domain, in which three protein-coding genes, i.e., Dlk1, Rtl1, and Dio3 are monoallelically expressed from the paternal allele, while multiple noncoding transcripts, such as Gtl2, Rtl1as, Rian, and Mirg, are maternally expressed. Active genes on each allele are represented by white rectangles with their transcriptional directions shown by arrows. IG-DMR is methylated (solid circle) and unmethylated (open circle) at the paternal and maternal alleles, respectively. Gtl2-DMR is a secondary DMR. The structure of mouse IG-DMR (mIG-DMR) sequences used in this study is enlarged beneath the map. C Structure of the 150-kb human β-globin locus YAC, in which LCR and β-like globin genes are indicated by shaded and solid boxes, respectively. Each of the mIG-DMR and rH19 ICR fragment was floxed by a pair of loxP variants [loxP5171 (solid triangle) and loxP2272 (open)], tandemly arranged and introduced 3' to the LCR for employing co-placement strategy. The expected SfiI restriction enzyme fragments (thick lines) generated from the YAC transgene and probe locations (filled rectangles) are shown beneath the map. D In vivo Cre-loxP recombination to derive mIG-DMR or rH19 ICR TgM. Recombination between two loxP5171 sites (solid) in the parental mIG-DMR/rH19 ICR transgene, for example, would generate rH19 ICR allele, during which one of the loxP2272 sites (open) is concomitantly removed to prevent further recombination. E Long-range structural analysis of the YAC transgene prior to Cre-loxP excision reaction. DNA from thymic cells was digested with SfiI in agarose plugs and separated by pulsed-field gel electrophoresis, and Southern blots were hybridized separately to probes. F Tail DNA from parental and daughter YAC-TgM sublines was digested with BglII (G: left) or DraI (D: right) and analyzed by Southern blotting using the probes shown in D (HS1-5' and HS1-3' probes for BglII and DraI digestion, respectively) to confirm correct recombination events