Production Of A New Generation Of Genetically Modified Pigs For Xenotransplantation

Objective:To produce new generation of genetically modified pigs as a better donor for xenotransplantation, CIITA-DN expression vector was transfected into DKO/CD46/TM porcine fetal fibroblast cells to obtain DKO/CD46/TM/CIITA-DN transgenic pigs. The class II transactivator (CIITA) is the master control factor of the SLA (MHC) II expression and the comprehensive regulator of antigen presentation. We expect the CIITA-DN will suppress porcine SLAII expression.Methods:A fragment of BGH-loxp-Zeo-Sv40pa-loxp with Zeocin resistance gene was inserted into the pMD-108T vector. The synthesized gene fragment was linked with the pST205vector which contained CIITA-DN gene by the particular restriction enzyme. A CIITA-DN transgene under a CMV β-actin promoter with an endothelium-specific Tie2enhancer was constructed and named as pNMU105. A porcine fetal fibroblast cell line (DKO/CD46/TM) was transfected with linerized pNMU105. Zeocin resistant colonies were isolated. PCR confirmed positive cells were used for nuclear transfer to clone pigs. All the piglets’genomes were extracted from their ear tissue for gene identification.Results:CIITA-DN expression vector was constructed. After transfection and antibiotic screening, we got dozens of Zeocin resistant colonies. The DKO/CD46/TM/CIITA-DN transgenic pigs were produced. The expected bands of592bp were visible in the results of PCR.Conclusion:Through the cultivation of a new generation of genetically modified pigs, the clone pig model was constructed that would theoretically suppress HAR and complement reaction, reduce coagulation and the human CD4+T cell immunity reaction. Thus we have made a new attempt to greatly improve the survival rate of transplanted xeno-organs in xenotransplantation.