抄録
Lack of alpha-1,3-galactosyltransferase (GTKO) and efficient expression of membrane cofactor protein (MCP) in pigs have been considered as a basic genetic platform for xenotransplantation. In addition, simultaneous expression of thrombomodulin (TBM) in the pig was reported to contribute extended survival of cardiac xeno-grafted monkey. The purposes of this study were to enhance human MCP and TBM expressions by using different promoters in single construct. A strategy was approached to enhance the expression level of MCP protein with codon modification. Construct CAGMCP was engineered using CAG promoter, and subsequently, TBM cDNA was directly ligated to its 3’ end with FMD 2A signal sequence, referred to as CAGMCP2ATBM. Additionally pIcam2TBM was constructed for TBM expression under control of porcine Icam2 promoter and then ligated to CAGMCP with reverse orientation, referred to as CAGMCPpIcam2TBM. The constructs were transfected into early passage ear fibroblasts originated from GTKO pig. The MCP-positive cells were separated 2 times by MACS cell sorter. The human MCP and TBM genes were transfected in all MCP-positive pig cell groups and MCP protein expressions were enhanced. On the other hand, TBM expressions were controlled by different promoters, showing high-level expression under CAG promoter, and endothelial cell-specific expression of TBM under pIcam 2 promoter. Finally, the cells expressing higher MCP compared GTKO cells was selected and could be applied for nuclear transfer as donor cells. Quantitative real-time RT-PCR and flow cytometry showed that all selected clones lead to the efficient expression of MCP. Taken together, MCP expression was successfully enhanced ubiquitously, while, TBM expression was controlled specifically in endothelial cells by Icam2 promoter, in a dual-expression vector.
