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.

The present study was designed to evaluate the short-term formula feeding effects on the in vivo embryo production with blood profiling and hormonal evaluation in cow. We have selected the cows randomly without consideration of estrous cycle and then divided the cows into two groups (control: 2.5 kg/day & without feeding group: 0 kg/day). The short-term formula feeding was provided 23 days and started for before 1 week of the insertion of CIRD in the vagina until the recovery of embryos. We have used the CIRD, FSH, PGF2α and GnRH for estrus synchronization and CIRD was removed before recovery of embryos. For analysis of blood profile, blood was collected before 1 week (D-7), insertion day of CIDR (D0), artificial insemination day (D8) and embryo collection day (D16) during experiment period. Our data demonstrated that, there were no significant differences in the percentage of superovulation between the control and without feeding group, however there was significant difference regarding the number of recovered embryos between donor control (9.6±1.1) and without feeding group (12.0±1.2) (p<0.05). Similarly, the number of transferable embryos in without feeding group (9.1±1.1, 76.0%) were also higher than control group (6.1±1.0, 64.0%) (p<0.05). There was no significant difference on the concentration of glucose, BUN (blood urea nitrogen) and cholesterol level among the groups. Furthermore, estradiol and progesterone level also has no significant differences between on control and without feeding group with normal range. In conclusion, provision short-term formula feeding will facilitate the in vivo embryo production efficiency of high yielding cows without any alteration of blood profiling.