(A,D,G) Allele-specific bisulfite analysis of Zac1 (A), Impact (D), and H19 (G). Each line represents the clone of an allele. Each circle represents a CpG dinucleotide where closed circles indicate methylation and open circles indicate no methylation. Maternal and paternal alleles are indicated. (B,E,H) Relative expression analysis of Zac1 (B), Impact (E), and H19 (H). Expression normalized to β-actin. Error bars indicate S.E.M. p-values calculated using an unpaired t-test with n.s. indicating p>0.05, * = p<0.05, ** = p<0.005, **** = p<0.0001. All asterisks indicate statistical significance. (C,F) Allele-specific expression of Zac1 (C) and Impact (F). The polymorphic base is highlighted in yellow. For Zac1, the maternal allele SNP is T (red) in highlighted position and paternal allele SNP is C (blue) in electrophoretogram. For Impact, the maternal allele SNP is A (green) in highlighted position and paternal allele SNP is G (black) in electrophoretogram. All analyses were performed on 2 staged matched B6/CAST hybrid control pups and 2 maternal effect progeny (MEP) exhibiting perinatal lethality.