We have attempted to produce identical genetic offspring in the rhesus monkey. Strategies have included the splitting of early- to mid-blastula stage embryos produced by IVF and dissociation of individual blastomeres from two-cell or four-cell stage embryos with their subsequent culture in vitro. We have now embarked upon the use of nuclear transplantation technology; the creation of enucleated oocytes and the isolation of individual blastomeres, or cells, from 16-32 cell stage embryos or from embryonic stem cells supplied by Dr. James Thomson (WRPRC) with their subsequent combination by electrofusion. The resultant embryos are studied for preimplantation developmental capability and will eventually be transferred to synchronized recipients in an effort to establish pregnancies. Oocytes derived from protocols involving recombinant hormones have been used in nuclear transplantation studies. IVF-produced embryos at the 8-16 cell state, or embryonic stem cells, have been used as our source of donor nuclei. Following enucleation by micromanipulation, oocytes are activated by exposure to cycloheximide. A donor blastomere is placed in the perivitelline space by micromanipulation, and electrofusion is utilized to fuse the membrances of the blastomere and the enucleated oocyte. This embryo is then cultured for evaluation of the success of the procedure. Cleaving embryos have been produced in high yield following the nuclear transplantation protocol itemized above utilizing 8-16 cell embryos as nuclear donors. We currently have conducted two embryo transfers without establishing a pregnancy and additional embryos resulting from nuclear transplantation are currently stored in the bank awaiting embryo transfer.
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