Introduction: Pigs have been proposed as a promising source of the global shortage of donor organ. However, there are several problems of immune rejection caused by differences between species. Several genetic combinations have been reported to try to solve this problem; however, additional research is needed on transgenic pigs. In this study, we developed multi-transgenic Yucatan miniature pigs using gene manipulation and somatic cell nuclear transfer (SCNT) to eliminate GGTA1, CMAH, iGb3s, and β4GalNT2 (quadruple knock-out; QKO) four pig genes that induce antigen-antibody reactions and express hCD46, hTBM, and hCD200 three human genes to regulate complement activity, blood coagulation, and phagocytosis.

Methods: We constructed four types of CRISPR-Cas9 vectors to include gRNA for each gene deletion ― GGTA1, CMAH, iGb3s, and β4GaINT2. Additionally, we constructed the pCAGGS-hCD46-2A-hTBM vector to simultaneously express the human complement regulatory gene hCD46 and blood clotting inhibitory gene hTBM. And then, constructed five vectors were transfected into PERV-C negative Yucatan miniature pig fibroblasts by electrophoration. QKO/hCD46/hTBM cloned piglets were produced through SCNT. We transfected the pLenti6-hCD200 vector for hCD200 expression into QKO/hCD46/hTBM pig’s fibroblasts. QKO/hCD46/hTBM/hCD200 transgenic cloned pigs were produced by SCNT. Transgenic cloned pigs were confirmed at the genomic DNA level through PCR and sequencing, and inserted gene expression was confirmed using aortic endothelial cells (AECs) by FACS analysis. Also, phagocytosis was analyzed through co-culture with human macrophage.

Results: We successfully produced three healthy QKO/hCD46/hTBM/hCD200 transgenic cloned pigs. PCR and sequencing analysis of genomic DNA derived from the umbilical cord showed that four genes were knocked out, and both pCAGGS-hCD46-hTBM and pLenti6-hCD200 vectors were inserted. By FACS analysis, we confirmed that xeno-antigens were removed, and hCD46, hTBM and hCD200 proteins were well expressed in AECs. Furthermore, it was confirmed that phagocytosis was significantly reduced in QKO/hCD46/hTBM/hCD200 (3.98 %) compared to wild-type (56.7 %) and QKO/hCD46/hTBM (35.2 %) by FACS analysis.

Conclusion: In this study, we successfully produced QKO/hCD46/hTBM/hCD200 multi-transgenic cloned pigs. The transgenic pigs which are not only deletion of gene related to antigen-antibody immune rejection but also inserted genes that regulating complement activity, blood coagulation, and macrophage-mediated immune rejection can reduce the immune response during pig-to-human xenotransplantation. Therefore, we expect that the multi-transgenic Yucatan miniature pigs will be useful source animal for xenotransplantation.