(A) Developmental imprinting pattern of Kcnq1. Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.