The chimera assay is a sensitive test for detecting in vivo exposure of the mammalian germ cell to ionizing radiation. This exposure results in a cell proliferation disadvantage that is expressed in cultured mouse preimplantation embryos. In the assay, 4-cell embryos from non-exposed parents are paired with 4-cell embryos from either exposed male or female parents to form aggregation chimeras. The assay's endpoint is a """"""""proliferation ratio"""""""", which is the number of cells contributed by the experimental partner embryo divided by the total number of cells comprising a chimera after 2-3 rounds of cell division. Ratios significantly less than 0.5 indicate than an experimental partner embryo expressed a cell proliferation disadvantage in the chimera. Progeny cells from the two partner embryos are currently distinguished by labelling the control partner embryo with the cytoplasmic dye FITC prior to chimera construction. However, label dilution from successive cell divisions precludes postimplantation analyses and heritability tests. This project's objective is to develop an 'enhanced' chimera assay to examine the effects of parental germ cell exposure in the male that persist beyond implantation and birth. Our First Specific Aim is to incorporate a heritable non-expressing transgenic cell lineage marker to replace the short-term cell lineage marker FITC in the chimera assay. The Second Specific Aim is to use our transgenic cell lineage marker to extend the chimera assay into postimplantation analyses of fetal and postnatal development. The Third Specific Aim is to apply the chimera assay with the transgenic cell lineage marker for heritability testing after parental exposure to reproductive toxicants.
