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.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of α1,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human EF1α promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.