In those previous experiments, where we also compared co-transferred IVP and nuclear transfer-generated embryos at similar stages, the proportion of embryos without an epiblast was one quarter for all types of embryos. To further verify this, we performed an additional round of NT using an EF5 cell line containing a construct with the LacZ reporter substituting the BAD gene. Nine out of twelve embryos retrieved contained an epiblast. We stained one such advanced embryo for b- Gal so as to monitor expression levels in different lineages when using the CAG enhancer/promoter employed for the BAD and LacZ overexpression constructs. Similar expression levels were seen in all lineages. We thus infer that the loss of the epiblast in BAD transgenic embryos is not a somatic cell transfer or differential expression artifact. We observed that some of the recovered transgenic embryos were slightly darker than their wild type counterparts. This could be caused by a defect in the trophoblast or underlying hypoblast. Embryos were therefore examined for gene expression differences in a TE marker expressed at this stage. ASCL2 is ideally suited for this purpose, as it is expressed maximally in the Day 13 to 14 TE. ASCL2 was expressed in all transgenic embryos at similar levels to the IVP-derived co-transferred controls. Overall higher ASCL2 levels in line 2 embryos appeared to be recipient related as the co-transferred wild type embryos exhibited similar high levels. We next investigated whether BAD overexpression affected the hypoblast. Wild type Day 14 embryos were treated with proteases to allow mechanical separation of trophoblast and hypoblast layers. Using these purified cellular preparations, we designed and tested cattle PCR primers for a range of candidate genes based on the mouse literature and our unpublished observations. We determined GATA4 and FIBRONECTIN to be optimal for this purpose with GATA4 being hardly detectable in the TE and FIBRONECTIN showing 40 fold greater expression in the hypoblast. Comparing trophoblast + hypoblast tissue of embryos with an embryonic disc to those without revealed highly significant gene expression differences for the two hypoblast markers, but no difference for the trophoblast marker. We have shown a selective effect of ubiquitous BAD overexpression on the early embryonic lineages.