The production of genetically identical animals by nuclear transfer carries importance for biomedical research because fundamental investigations into the nature of the immune system become feasible. This proposal directly addresses one of the objectives of this PA by developing methods to produce clonally-derived populations of macaques. The Principal Investigator has established a successful rhesus monkey in vitro fertilization (IVF) program at the Oregon Regional Primate Research Center, where monkeys have been produced by nuclear transfer technology employing enucleated oocytes as recipient cytoplasts and individual blastomeres from IVF-produced embryos as nuclear donors. This proposal is designed to improve the nuclear transfer procedure in rhesus macaques by identifying a source of donor nuclei which can be used to support the creation of large clone sizes. A goal of this effort is to make available clonally-derived animals to investigators actively engaged in vaccine development and pathogenesis studies.
Specific aims i nclude: (1) to optimize the donor nuclear source for the production of clonally-derived animals. Three potential sources of nuclei will be evaluated - blastomeres of IVF embryos (a method demonstrated as successful), primary cultures of fibroblasts, and immortalized embryonic stem (ES) cells. Initially, nuclei from fetal and adult fibroblasts and ES cells, transfected with green fluorescent protein to allow confirmation of donor nucleus participation in development, will be examined for their ability to support preimplantation development following nuclear transfer into metaphase II cytoplasts. Experimentation will determine whether cell cycle staging is critical for successful reprogramming of the nucleus following transfer; and (2) to produce clonally-derived rhesus monkeys. The optimal source of donor nuclei established under Aim 1 will be employed to produce clones of 50 reconstituted embryos. These will be cryopreserved using techniques demonstrated in the Principal Investigator's laboratory. Transfers involving two embryos of the same clone will be conducted in synchronized host mothers. IVF-produced embryos will be employed as a last resort in the event that conditions for fibroblasts or ES cells cannot be established.
| Nusser, K D; Mitalipov, S; Widmann, A et al. (2001) Developmental competence of oocytes after ICSI in the rhesus monkey. Hum Reprod 16:130-137 |
