The objective was to successfully reconstruct preimplantation blastocyst stage mouse embryos. This process, referred to as blastocyst reconstitution, attempts to circumvent fetal rejection associated with interspecies embryo transfer. Development of practical and efficient procedures for isolating the precursor placental (trophectoderm) and fetal (inner cell mass) cellular components of the developing blastocyst were the principle goals of this project. By injecting the inner cell mass of one species (or genotype) into the trophoblast (trophectodermal vesicle) of a genetically dissimilar recipient, it may be possible to minimize fetal loss associated with immunological rejection. Thereby, the reproductive potential of a genetically valuable animal (e.g., rare animal model or endangered species) is increased. Also, the overall utility of the NIH Animal Genetic Resource is enhanced. These experiments focused on whether it was possible to (1) use heterologous antibodies to isolate the inner cell mass immunosurgically, (2) selectively irradiate and destroy blastomere of the inner cell mass to form zona pellucida-intact trophectodermal vesicles, and (3) inject cellular components into mouse blastocysts and zona-intact trophectodermal vesicles, at 5 degrees C, using a large diameter (30 micron) pipette in a two-microtool procedure. Results demonstrated that xenogeneic antibodies bound to trophectoderm surface antigens and mediated immunosurgical ICM isolation, although the lytic response varied with antibody source. Chemical approaches for production of trophectodermal vesicles were less effective than irradiation. Additionally, holding blastocysts at a lower temperature (5 degrees C) permitted penetration of large pipettes which was not detrimental to embryo viability.
