FIG. 5.

Normal imprinting at IC2 in reciprocal DelTel7 heterozygotes. (A) Diagram of the intron 10 region of Kcnq1, showing the position of IC2 (KvDMR1, white rectangle) (26), the structure of a 335-bp sequence containing the 31 CpG sites analyzed by bisulfite sequencing, and the region 4 fragment (R4) amplified by RT-PCR in panel B. Reciprocal DelTel7 heterozygous embryos were recovered at E9.5 from crosses between +/DelTel7 and (129CAST)F₁ mice. Genomic DNA samples were analyzed by sodium bisulfite mutagenesis and sequencing. The entire IC2 subdomain is deleted in the DelTel7 allele such that mutant embryos are hemizygous for the wild-type IC2 CAST allele. The results show normal differential methylation of the paternal (pat; unmethylated) and maternal (mat; methylated) CAST alleles of IC2 in these embryos. Methylated CpG sites are represented by filled circles, and unmethylated sites are indicated by open circles. Omitted sites indicate sequencing ambiguities. (B) Allele-specific expression of Kcnq1ot1 in DelTel7 embryos. The expression of Kcnq1ot1 was assessed by RT-PCR (region 4 [R4] in panel A) (68) on embryonic RNA samples collected at E9.5 from reciprocal crosses between +/DelTel7 and (129CAST)F₁ mice. For each embryo, the maternally (mat) and paternally (pat) inherited alleles are shown. Δ, Deltel7; +, wild type. Embryos with a wild-type CAST allele are numbers 1, 2, and 5 (pat CAST allele); numbers 6 and 7 have the mat CAST allele. RT-PCR for the housekeeping gene G3pdh was used as a positive control.