Abstract: 565

IN VITRO DEVELOPMENTAL POTENTIAL OF NUCLEAR TRANSFER (NT) EMBRYOS FROM THE RHESUS MONKEY.

Shoukhrat Mitalipov¹ , Kevin Nusser¹ , Andrea Widmann¹ , Behzad Gerami¹ , Don Wolf^{1 *}
Oregon Regional Primate Research Center, Beaverton, OR ¹

Recent evidence from several mammalian species indicates that cloned animals can be produced from embryos containing nuclear material donated from somatic cells. Our objective was to determine the in vitro developmental potential of NT embryos produced from fetal fibroblasts (FF) as a prelude to cloning efforts in the monkey. To this end, mature, metaphase II oocytes from superovulated monkeys were enucleated by removing the polar body and metaphase plate with epifluoresence confirmation of Hoechst stained DNA. Cytoplasts were exposed to cycloheximide, injected with FF (derived from a day-130 monkey fetus and serum starved 5-10 days prior to use) into the perivitelline space and fused by electroporation. Embryos were co-cultured on buffalo rat liver cells in CMRL + 10% FBS medium. For comparison, MII cohorts were fertilized by intracytoplasmic sperm injection (ICSI). Embryos were cultured for 10 days with co-cultures changed every other day. Overall, 8 out of 26 cleaved NT embryos (n=3 matched experiments) developed to compacted morulae (CM) and, of these, 3 developed to blastocysts (B). Compared to ICSI embryos, the development of NT embryos to CM and B was decreased (31% versus 76% for CM and 12% versus 44% for B; NT and ICSI respectively; p<0.05). However, the developmental rate was similar between groups (CM detected between days 5 and 6 of culture; B detected between days 7 and 9). To our knowledge, this is the first direct evidence that monkey NT embryos from differentiated cells follow normal development rates in vitro albeit at a lower incidence. These results suggest that NT technology can be used to generate genetically identical monkeys. Supported by NIH grants RR12804, A142709, PO1A136353, RR00163, HD18185 and Ares Serono.

    This abstract is being presented on Tuesday, August 3 at 5:15 PM at Todd 133.