The production of genetically-identical animals by nuclear transfer is of great importance to biomedical research, as it eliminates genotypic variation during experimental manipulation and allows greater statistical validity with fewer animals. We established a successful in vitro fertilization (IVF) program at the Oregon Regional Primate Research Center where rhesus monkeys have been produced for the first time by nuclear transfer. The technology employs enucleated, metaphase II oocytes as recipient cytoplasts (MII cytoplasts) and individual blastomeres from IVF-produced embryos as nuclear donors. Ongoing research is designed to improve the nuclear transfer procedure in rhesus macaques with the goal of making genetically-identical animals available to the biomedical community for experimental use while evaluating the efficiency and cost-effectiveness of this evolving approach. The three specific aims are 1. To optimize the preparation, maturation, activation and low temperature storage of MII cytoplasts. Conditions for the cryopreservation of MII cytoplasts will be established and in vitro maturation conditions will be sought to take advantage of the potentially large numbers of ovarian oocytes; 2. To establish a source of totipotent (potential to differentiate into all cell types present in the adult) embryonic cells or cultured embryonic stem cells (ES) as nuclear donors. Stem cell lines will be created from in vivo produced blastocysts and stably transfected with a molecular marker, such as green fluorescent protein. The totipotency of these lines will be evaluated in reconstituted embryos following nuclear transfer. Additionally, inner cell mass cells from IVF-produced embryos will be evaluated as nuclear donors and as a source of ES cell lines; and 3. To produce rhesus monkeys by conventional or serial nuclear transfer and evaluate blastomere separation and culture as an alternative method of cloning. We will produce 5 sets of genetically identical animals in the initial year and more thereafter by nuclear transfer. Also, the simplicity of separating IVF-produced embryos at early cleavage stages with the transfer of individual blastomeres or groups of blastomeres to surrogate zonae offers a low technology method of cloning that will be evaluated.
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