Fig. 5

Genomic imprinting is recapitulated in LCb478 YAC-TgM. A–C Two pairs of LCb478-TgM (2578 vs 2574 and 2607 vs 2612, 1 month old), each inheriting the transgene either paternally (P) or maternally (M) were made anemic and spleens were removed, from which one-quarter each was used for genomic DNA or total RNA preparation with the remaining half used for chromatin preparation. A DNA methylation status of the transgene was determined by Southern blot analysis using BamHI with (+) or without (−) BstUI (vertical lines) and a probe. *: parental or methylated, uncut fragments. B ChIP analysis of CTCF occupancy at the transgene. 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 LCb478), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S. D.), determined by three reactions, are depicted, as a signal for Necdin (P) was arbitrary set at 1.0. C The relative expression levels of the human β-globin gene, after normalization to that of the endogenous mouse α-globin gene were determined by RT-qPCR analysis. The average and standard deviation (S.D.), determined by three reactions, are depicted, as a value of No. 2612 animal was arbitrary set at 1.0. D Schematic representation of the genomic imprinting recapitulated in the transgene