In this study, we examined whether Hanganutziu‐Deicher (H‐D) antigens are important as an immunogenic non‐a1,3‐galactose (Gal) epitope in pigs with a disrupted a1,3‐ galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The a1,3‐galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote a1,3‐galactosyltransferase gene knockout (GalT‐KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of a1,3‐ galactosyltransferase activity when compared to those of control. Enzyme‐linked lectinosorbent assay showed that the heterozygote GalT‐KO pig had more sialyla2,6‐ and sialyla2,3‐ linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT‐KO pig had a higher N‐glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT‐KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. In this study, a pig GV oocyte extract (pGV extract) was developed. Treatment of pig fibroblasts with the pGV extract promoted colony formation after 2–3 weeks in culture, concomitant with the expression of stem cell markers (Oct‐4, Rex1, Nanog, Sox2) and repression of differentiated cell markers (CKAP2, NPR3 ). Using fibroblasts transfected with human Oct‐4 promoter‐driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4 ‐ EGFP cells by 10 days post‐treatment. These transgenic donor cells were injected into 8‐cell embryos. Oct‐4 promoter activity was subsequently detected in most ICM cells of the host blastocyst. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐ EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Additionally, the pGV extract promoted somatic cell reprogramming and cloned embryo development when assessed by measuring histone H3‐K9 hypomethylation, the expression of Oct4 and Nanog in blastocysts, and the production of increased numbers of high‐ quality blastocysts. Under specific culture conditions, pGV extract‐treated fibroblast cells differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig.