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Fig. 5. Expression of imprinted genes in placentas as a consequence of the IG-DMR deletion. (A) Northern blot analysis for imprinted genes in the Dlk1-Dio3 domain (Gtl2, Dlk1, Rtl1, Dio3), and primer extension assay (for micro-RNAs miR-127 and miR-410). MBII-48, MBII-49 and MBII-78 are snoRNA genes. Gapdh and U3 (Rnu3 - Mouse Genome Informatics) are controls. Gene symbols in blue and red denote those expressed from the paternally and maternally inherited chromosomes, respectively. Each genotype is represented by duplicate tracks of RNA isolated from different placentas. (B) Bar chart comparing expression levels between E16 placentas carrying the maternally-derived IG-DMR deletion (red; KO/+), with the paternally derived deletion (blue; +/KO), and wild-type littermates (yellow; +/+). Error bars represent s.e.m. Values were calculated from data generated for each gene using control (n=5-8) and mutant (n=2-5) placentas from different litters (n=2-4). Statistically significant differences from normal are: ^*, P<0.05; ^**, P<0.005; ^***, P<0.0005; ANOVA + Fisher's PLSD test. Values were normalised against Gapdh except the small RNAs which were normalised against the unlinked, non-imprinted snoRNA U3. For comparison, the inserted panel shows the equivalent expression analysis in E16 embryos (see Lin et al., 2003). (C) Biallelic expression of Dlk1 in placentas of conceptuses with a maternally transmitted IG-DMR deletion. Sequence analysis of RT-PCR products from control littermate and heterozygote placenta upon maternal transmission of the IG-DMR deletion. Activation of Dlk1 from the normally repressed maternally inherited allele is only observed when the IG-DMR deletion is maternally transmitted. The Dlk1 expression in the normal placenta is exclusively from the paternal allele.

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