The preparation of fetal fibroblasts and eGFP gene infection were conducted as previously reported. Fetal fibroblasts were obtained from a 35-day fetus and cultured in the same conditions that were used for the ear skin fibroblasts. To infect the cells, a replication-defective vector based on Moloney murine leukemia virus, pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) was used. Retroviral vector pseudotyped with VSV-G was carrying an eGFP gene under the control of the cytomegalovirus (CMV) promoter, LNCE-(VSV-G), which was kindly provided by Dr. A.W.S. Chan. LNCE had long terminal repeat, neomycin-resistant gene, CMV promoter, and eGFP. Cells were infected with the retroviral vector by the following methods. Polybrene (0.1%) was diluted 1:30 with 0.1 X HBS medium (22.9 mM Hepes, 140.3 mM NaCl, 0.7 mM NaH2PO4H2O). Three hundred microliters of the diluted medium were added to 4 |xl of vector solution (108 cfu/ml). The solution was diluted in 5 ml culture medium and incubated overnight. G-418 selection was started the following day, it continued for 13 days, and then the cells were frozen. The cells underwent this process up to seven times. The cells were thawed, cultured, and then serum-starved (0.5% serum) for 3-5 days before NT. This is the same cell line that was reported by Park et al. that resulted in five cloned piglets and has resulted in a single cloned piglet when synchronized in G2/M before NT (unpublished).
After 42 to 44 h of culture, oocytes were freed from cumulus cells by vigorous vortexing for 4 min in TL-Hepes supplemented with 0.1% PVA and 0.1% hyaluronidase. Cumulus-free (denuded) oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm in enucleation medium with a glass pipette 30 |xm in diameter. A single donor cell was placed in the perivitelline space of the oocyte to contact the oocyte membrane.
Fusion/Activation of Oocytes
Injected oocytes were placed between two 0.2-mm diameter platinum electrodes 1 mm apart in activation medium. Fusion/activation was induced with two successive DC pulses of 1.2 kV/cm for 30 |xsec on a BTX Elector-Cell Manipulator 200 (BTX, San Diego, CA). Nonmanipulated oocytes were electrically activated by using the same pulse parameters and cultured as controls.