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.

In our previous studies, the cardiac xenotransplantation from an alpha-1,3-galactosyltransferase knockout pig (GT-MCP-MCP) to cynomolgus monkeys showed a mean survival of 38 days. The objective of this study is to genetically upgrade the GT-MCP-MCP pig, to further enhance membrane cofactor protein (MCP) expression and to express an endothelial specific thrombomodulin (TBM). MCP is a complement regulatory protein and TBM is a coagulation inhibitor. As the dicistronic cassette for wild-type-based MCP and TBM concurrent expressions does not show any increase of MCP, we optimized the MCP codon usage (mMCP) and substituted mMCP for MCP. When the mMCP-TBM cassette was transfected to HeLa cells, we were able to find an increased expression of MCP and endothelial cell-specific TBM expression. The cassette was then transfected into ear-skin fibroblasts isolated from one-month-old #23-4 of a GT-MCP-MCP pig, and the cell populations expressing MCP were obtained by MACS cell sorting. We performed a single cell culture of the selected cells, and obtained clones over expressing 90% MCP. The cells of a clone were used as a donor for nuclear transfer and generated GT-MCP/-MCP/mMCP/TBM pig. The transgenic pig was confirmed to be carrying the cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Thus, we believe that the GT-MCP/-MCP/mMCP/TBM transgenic pig would be potential for the prolongation of xenograft survival in the recipients.

The current study was conducted to evaluate the biocompatibility of α-1,3 galactosyltransferase knockout pig bone graft in a rat calvarial defect model. Porcine cancellous bones were harvested from general and alpha-gal KO pigs and washed with 70% ethanol solution and normal saline. Bone pieces of the alpha-gal KO pig underwent a chemical treatment process to delipidize and deproteinize the bone. Bone graft particles were freeze-dried and stored at −70°C until use. Each bone graft was implanted into the rat calvarial defect in a fresh general pig, fresh transgenic pig, and chemical-treated pig bone group. There was no systemic adverse effect on hematology or necropsy findings in all groups at 1 week and 4 weeks. In the microcomputed tomography analysis, bone volume increased significantly in the chemical-treated transgenic pig bone group, whereas bone mineral density decreased significantly in the fresh general pig bone group compared with other groups. Histological evaluation showed cellular infiltration located at the margin of the bone graft particles, especially in the fresh general pig bone group. These results indicate that fresh general pig bone can elicit a greater local inflammatory response than fresh transgenic pig bone. Further, chemical-treated transgenic pig bone graft was less immunogenic than fresh bone graft. In conclusion, transgenic pig bone is a more biocompatible graft material. In addition, chemical treatment can reduce bone graft immunogenicity by delipidizing and deproteinizing bone.