The medium used for oocyte maturation was tissue culture medium (TCM) 199 (31100035; Gibco, Grand Island, NY) supplemented with 0.1% polyvinyl alcohol (PVA), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 |xg/ml LH (L-5269; Sigma Chemical Company, St. Louis, MO), 0.5 |xg/ml FSH (F-2293; Sigma), 10 ng/ml epidermal growth factor (E-4127; Sigma), 75 |xg/ml penicillin G, and 50 |xg/ml streptomycin.
The medium used for enucleation was TCM 199 supplemented with 0.3% BSA (A-8022; Sigma) and 7.5 |xg/ml cytochalasin B, and the medium for injection was the same medium, but without cytochalasin B. The medium used for activation consisted of 0.3 M mannitol, 1.0 mM CaCl2H2O, 0.1 mM MgCl2-6H2O, and 0.5 mM Hepes.
The medium used to culture reconstructed embryos was North Carolina State University-23 medium supplemented with 0.4% BSA.
Collection and Culture of Cumulus-Oocyte Complexes
Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl solution at 35-39°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (2-6 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe. COCs were washed three times in maturation medium, and 50-60 COCs were transferred to 500 |xl of the same medium that had been covered with mineral oil in a 4-well multidish (Nunc, Roskilde, Denmark) and equilibrated at 39°C in an atmosphere of 5% CO2 in air overnight.
Preparation of Ear Skin Fibroblasts and Fetal Fibroblasts
A small ear skin biopsy was obtained from the transgenic pig 402-2 at 4 days of age, and the tissue was cut into small pieces with fine scissors. The cells were incubated for 30 min at 37°C in PBS containing 0.05% trypsin and 0.5 mM EDTA, and this suspension was centrifuged. The cell pellet was resuspended and cultured in Dulbecco modified Eagle medium supplemented with 75 |xg/ml penicillin G, 50 |xg/ml streptomycin, and 15% (v:v) fetal calf serum. The cells underwent this process up to four times. They were thawed, cultured, and then serum-starved (0.5% serum) for 3-5 days before NT (Fig. 1).