Generation Of Transgenic Expression Human CD47 Pig And The Role In Attenuating Macrophage-mediated Xenograft Rejection

Xenotransplantation is an effective solution for resolving the problem of inadequate supply of donor organs. Pigs were considered the ideal donor for transplantation to human recipients. However, xenograft would be cleared by host macrophages after entering the receptor because that the SIRPα receptors on macrophages couldn’t interact with the surface molecule CD47 on xenograft cells in a species-specific manner. It would lead to the xenograft cells were cleared by host macrophages. This study was to generate the transgenic expression human CD47 gene pigs on the basis of α-1,3-galactosyltransferase gene knockout pigs(GTKO) and that could avoid hyperacute rejection and weaken the phagocytosis by host macrophage.Firstly p CAGGS-h CD47-Neo vector to expressing the h CD47 gene under the CMV 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 h CD47 gene expression in transgenic pig’s heart, liver, spleen, lung, kidney and pancreas was analyzed by q RT-PCR, Western Blot and immunohistochemistry. We detected the haemal physiological parameters by drawing blood from the survival piglets to monitor health condition. The porcine bone marrow mesenchymal stem cells were isolated from bone marrow by gradient centrifugation, and cellular surface antigen CD29、CD34、CD44、CD45 and CD90 expression were examined by flow cytometry. CFSE-labeled porcine peripheral blood mononuclear cells were incubated with human macrophages for 4h or 8 h at 37°C, and phagocytosis of CFSElabeled targets were measured by FCM analysis. The results were detailed as follows:1. Firstly p CAGGS-h CD47-Neo vector to expressing the h CD47 gene under the CMV early enhancer/chicken β actin promoter(CAG) was constructed and verified by enzyme digestion and sequencing test. The results showed that the components of p CAGGSh CD47-Neo sequence were corrected. And use the vector to transfection fibroblasts in vitro, and the expression of RNA and protein levels were detected by RT-PCR and Western Blot after 24 h. The results showed h CD47 gene in ear fibroblasts has obvious expression of RNA and protein. 2. The ear fibroblasts of GTKO Bama miniature pig were transfected by p CAGGSh CD47-Neo. There were 46 single cells cloning. 647 numbers of embryos, which reconstructed by somatic cell nuclear transfer, were transplanted in 4 numbers of receptor. The transgenic cloned piglets were identified by PCR. The results showed that 13 transgenic cloning piglets were all h CD47 gene positive expression. 3. The h CD47 gene expression in transgenic pig’s heart, liver, spleen, lung, kidney and pancreas was analyzed by q RT-PCR. The results showed that h CD47 gene had the highest expression level in pancreas, followed by liver, lung, spleen, kidney and the lowest expression level in heart. 4. The heart, liver and pancreas tissue of transgenic cloned piglets had detected by immunohistochemistry and Western Blot. The results showed that the h CD47 gene expression in transgenic pig’s pancreas and liver tissue. 5. We detected the haemal physiological parameters by drawing blood from the survival piglets to monitor health condition. The results showed that the haemal physiological parameters of transgenic cloned piglets were normal, and compared with the wild type Bama miniature pig had no significant difference(p > 0.05).The haemal physiological parameters showed that survival piglets were in good health. 6. The p MSCs cellular surface antigen CD29 、 CD34 、 CD44 、 CD45 and CD90 expression were examined by flow cytometry. The results showed that p MSCs cells expressed CD29, CD44 and CD90, no expression of CD34 and CD45. 7. The h CD47 genes expression in p MSCs and PBMC were detected by flow cytometry. The results showed that the h CD47 gene expression in PBMC and p MSCs. 8. The macrophages from THP-1 cells were incubated with PBMC in vitro showed that expression h CD47 PBMC survival rate was significantly higher than not expression h CD47 PBMC(p < 0.05).All above, this study had successfully generated the GTKO Bama miniature pig that expressing h CD47 genes. Expression model of h CD47 in porcine organs was identified, that would help study the role of h CD47 gene weakening the host macrophages phagocytosis of different kinds of xenograft organs.