Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of α-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

The objective of this study was to investigate the effects of oxygen tension during in vitro maturation of porcine oocytes on the nuclear maturation and differences in gene expression. Cumulus-oocyte complexes (COCs) were collected from ovaries obtained at a local slaughterhouse, matured for 44 hours in TCM199 supplemented with porcine follicular fluid (pFF) under 5% or 20% oxygen concentration. In results, oxygen tension had no significant effects on nuclear maturation. Relative poly(A) mRNA abundance of MnSOD, CCNB1, LDHA, G6PD, BCL, GPX1, IGFR2, GLUT1, BAX, GREM, PTGS2 was analysed in cumulus cells. GLUT1, G6PD, LDHA were up-regulated in the cumulus cells matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas CCNB, MnSOD were up-regulated in the cumulus cells matured in high oxygen, which suggest a higher activity of mitosis-promoting factor and antioxidant response. In conclusion, cumulus cells increase in glucose metabolism via anaerobic glycolysis under low oxygen concentration and show significant change in antioxidant against oxidant damage or apoptotic response under high oxygen concentration. For such an effect of cumulus cells, oocytes could be matured normally regardless of various oxygen concentration.