Porcine Somatic Cell Nuclear Transfer

SCNT was performed as previously described [23] (link), [25] (link). After culturing for 38 h to 42 h, oocytes with expanded cumulus cells were briefly treated with 0.1% (w/v) hyaluronidase and denuded of cumulus cells using a finely drawn glass capillary pipette. Oocytes extruding the first polar body with uniform cytoplasm were cultured in NCSU23 medium supplemented with 0.1 µg/mL demecolcine, 0.05 M sucrose, and 4 mg/mL bovine serum albumin (BSA) for 0.5 h to 1 h. The oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette (approximately 20 µm in diameter) in Tyrode’s lactate medium supplemented with 10 µM hydroxyethyl piperazineethanesulfonic acid (HEPES), 0.3% (w/v) polyvinylpyrrolidone, and 10% FBS in the presence of 0.1 µg/mL demecolcine and 5 µg/mL cytochalasin B. Any protrusion observed on the surface of an oocyte was removed along with the polar body. Fetal, newborn, and adult fibroblasts of the fourth to ninth passages were used as nuclear donors after cell cycle synchronization by 0.5% FBS serum starvation for 48 h. A single donor cell was inserted into the perivitelline space of an enucleated oocyte.
Donor cells were fused with the recipient cytoplasts with a single direct current pulse of 200 V/mm for 20 µs using an embryonic cell fusion system (ET3, Fujihira Industry Co. Ltd., Tokyo, Japan) in fusion medium [0.25 M d-sorbic alcohol, 0.05 mM Mg(C₂H₃O₂)₂, 20 mg/mL BSA, and 0.5 mM HEPES (free acid)]. The reconstructed embryos were cultured for 2 h in PZM-3 and then activated with a single pulse of 150 V/mm for 100 µs in an activation medium containing 0.25 M d-sorbic alcohol, 0.01 mM Ca(C₂H₃O₂)₂, 0.05 mM Mg(C₂H₃O₂)₂, and 0.1 mg/mL BSA. The reconstructed embryos were equilibrated in PZM-3 supplemented with 5 µg/mL cytochalasin B for 2 h at 38.5°C in humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ (APM-30D, ASTEC, Japan).