Testing for transgenerational epigenetic inheritance of the aberrant imprinted expression. (A) Breeding design to test whether ED-perturbed parental allele-specific transcription is transgenerationally inherited through the paternal germline to an unexposed generation. G1 male fetuses were exposed in utero to EDs or vehicle control (‘oil’) daily from 12.5 dpc to 16.5 dpc. After reaching adulthood, 129S1 G1 males were mated with 129S1 unexposed females to generate G2 offspring (3 blue stars), which derived from exposed prospermatogonia. At adulthood, G2 males were mated with unexposed JF1 females to generate G3 offspring, which were never directly exposed to EDs. JF1 × 129 G3 fetuses were dissected at 13.5 dpc to collect organs for RNA isolation. Parental-specific transcription was quantified in the total RNA using multiplex SNuPE assays. (B) Results of Sequenom allelotyping experiments using heart and lung tissue of the G3 generation; color scale as in Figure 2; letters in parentheses denote independent SNPs. Notice the lack of inherited changes from the exposed generation. More groups of fetuses are shown in Additional file 2. This Figure includes standards that are routinely included in the Sequenom runs (see Methods).