Porcine nuclear transfer (NT) was performed as described by Park et al.. Briefly, the cumulus-free oocytes were enucleated in TCM/BSA medium supplemented with 7.5 |xg/ml of cytochalasin B. The first polar body and the surrounding cytoplasm were removed with the aid of a beveled pipette (inner diameter, —25-30 |xm). Enucleated oocytes were kept and micromanipulated in TCM/BSA until NT. A Nikon Diaphot (Nikon Corporation, Tokyo, Japan) equipped with a 40X objective and a pair of Narishige micromanipulators (Narishige International USA, East Meadow, NY) was used for micromanipulations. Donor fibroblasts (fetal fibroblasts were isolated from a 35-day-old pig fetus) were selected according to their size and shape (small cells with smooth membranes).
A single cell was transferred into the perivitelline space with the same pipette as used for enucleation. Cytoplast-fibroblast complexes were placed between two electrodes (1 mm apart), overlaid with fusion medium (0.3 M mannitol, 1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes), and aligned manually. Fusion/ activation was achieved by two pulses of 1.2 kV/cm for 30 |xsec as measured by the BTX-optimizer (BTX, San Diego, CA). Fused embryos were cultured for up to 6 days in 500 |xl of NCSU-23 supplemented with 4 mg/ ml of BSA.