Abstract：Xenotransplantation is an effective solution for resolving the problem of inadequate supply of donor organs. Pigs (Sus scrofa) are considered the ideal donor for transplantation to human (Homo sapiens) recipients. However, xenograft would be cleared up by the host macrophages after entering into the receptor. The reason is that the singal regulatory protein α (SIRPα) receptors on macrophages can't specifically recognize the surface molecule integrin associated protein (CD47) on xenograft cells. This study was to generate α-1, 3-galactosyltransferase gene (GTKO) knockout pigs expressing hCD47 which could avoid hyperacute rejection and weaken the phagocytosis by host macrophage. Firstly pCAGGS-hCD47-Neo vector to expressing the hCD47 gene under the cytomegalovirus early enhancer/chicken β actin promoter (CAG) was constructed. The ear fibroblasts of GTKO Bama miniature pig were transfected. Then the transgenic cloned piglets were generated by somatic cell cloning techniques. The transgenic cloned piglets were identified by PCR. The hCD47 gene expression in transgenic pig's heart, liver, spleen, lung, kidney and pancreas was analyzed by qRT-PCR, Western blot and immunohistochemistry. The haemal physiological parameters were detected by drawing blood from the survival piglets to monitor health condition. The results showed that 13 transgenic cloning piglets were all hCD47 gene positive expression. The hCD47 gene had the highest expression level in pancreas, followed by liver, lung, spleen, kidney and the lowest expression level in heart. Western blot and immunohistochemistry results also showed that expression of the hCD47 gene was strong in the pancreas and liver, consistent with the results of qRT-PCR. The haemal physiological parameters showed that survival piglets were in good health. All above, this study had successfully generated the GTKO Bama miniature pig which expressed hCD47 genes. Expression model of hCD47 in porcine organs was identified, which would be helpful for studying the role of hCD47 gene in weakening the phagocytosis of host macrophages for various xenograft organs.
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