Production of transgenic porcine blastocysts by nuclear transfer

In this study the in vitro development of porcine nuclear transfer (NT) emb ryos was investigated. Transgenic fetal fibroblast cells that were frozen a fter 5 days of serum starvation were injected immediately after thawing int o enucleated metaphase II (MII) oocytes. Reconstructed embryos were activat ed by incubation in 200 mu M thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented wit h 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing greater than or equal to 2 nuclei) in 6 replicates was 33% (ranging from 15% to 50 %). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. Th e blastocyst rate was significantly lower for NT embryos as opposed to part henogenetically activated embryos (1% vs 5%; P < 0.05). The neomycin-resist ance gene was amplified by PCR in all three NT embryos, indicating their or igin from the injected transgenic fibroblasts. Efforts ave now being direct ed in improvements in the nuclear transfer technology, whereby viable fetus es or offspring can be produced from these NT-embryos. Mel. Reprod. Dev. 56 :145-148, 2000. (C) 2000 Wiley-Liss, Inc.