Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum.

In this study, we attempted to upgrade pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized () and () designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected into ear-skin fibroblasts isolated from a pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.