Abstract

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybrl4 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomédical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.