Fig. 6

Genomic imprinting recapitulated in the LCb118 YAC-TgM. a–c Two pairs of LCb118-TgM (ID No. 2601/2602 and 2633/2640), each inheriting the transgene either paternally (P) or maternally (M) were made anaemic 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 λ probe. *: methylated, uncut fragments in BstUI (+) lanes. 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 LCb118 transgene), 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 (M) was arbitrary set at 1.0. Statistical differences were determined using an unpaired t test (N.S., not significant). 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. 2640 animal was arbitrary set at 1.0