Generation Of Expressing Human Complement Regulating Protein Xenotransplantation Donor Pig

Pigs are excellent donors for xenotransplantation,but the immune rejection that occurs in xenotransplantation is a difficult problem to be solved.The hyperacute reaction(HAR)of pig to human organ transplantation caused by ?-1,3 galactosidase(Gal)has been solved basically.hCD55,hCD46,which are highly efficient expression in complement regulatory genes,could reduce effectively the immune rejection in xenotransplantation.In this study,random integration methods were used to transfer human decay accelerating factor hCD55 gene,on the basis of ?-1,3-galactosyltransferase gene knockout(GTKO)and human membrane co-factor protein hCD46 transferred from the scrofa(Sus scrofa)ear fibroblasts,obtained one cloned pigs which is hCD55 transgenic pig.And the site-specific was also used to transfer hCD55 gene,by utilizing GTKO Bama minipig ear fibroblasts.In order to generate expressing efficiently h CD55 ear fibroblasts cell lines by hCD55 site-specific intergation.The specific findings are as follows:1.Generation of hCD55 and hCD46 double complement transgenic cloning pigsIn this study,we used the GTKO/hCD46 Bama-wuzhishan hybrid minipig ear fibroblast cell line,to generate the hCD55 expressing clone pig.construct a gene expression vector pCAG-hCD55,which is widely expressed in the CAG-regulated hCD55 promoter,and transfected into the porcine fibroblast cell line.The puromycinselected positive cells 52.The PCR method was used to identify the transgenic pigs.The number of positive clone cell is 27.RT-PCR was used to identify the expression of hCD55.The number of hCD55 expression clones are 17.Somatic cell nuclear transfer technology to generate hCD55 transgenic pigs.and the expression of hCD55 was analyzed in various organs of transgenic pigs by qPCR.A total of hCD55 positive piglets were obtained.qPCR detected the hCD55 gene in the piglets heart,liver,spleen lungs and kidneys.It is expressed in organs such as pancreas,pancreas,muscle,small intestine,and large intestine,and is expressed higher in liver,lung,and kidney.This study successfully generated hCD55 transgenic pigs,and hCD55 gene has a certain level of expression in the main organs of piglets,providing a good donor for xenotransplantation research.2.Establishment of Knock-in Loxp-modified Human(Homo sapiens)CD55 Gene Fibroblast Cell Line with Porcine(Sus scrofa)Rosa26 LocusEfficient expression of human complement regulatory gene hCD55 is particularly important in xenotransplantation.Human CD55 gene was knocked-in porcine Rosa26(pRosa26)locus via CRISPR/Cas9 on the basis of GGTA1 knockout(?-1,3-galactosyltransferase knockout,GTKO)swine ear fibroblasts,to establish the cell lines with efficient expression of hCD55 gene.Generating a Cas9 expression vector targeted the intron 1(intron 1)of the reverse orientation splice acceptor ?-geo26(Rosa?geo26)site in swine.A expression vector homologous-recombinating hCD55 at the pRosa26 site were constructed,which contained the the hCD55 gene under the control of polypeptide chain elongation factor 1?(EF1?)in the heterogeneous Loxps.The hygromycin B phosphotransferase(HPH)gene was the positive screening gene.The thymidine kinase(TK)was the negative gene.The Cas9-Rosa26 expression vector and the pEF1?-hCD55 expression vector were co-electroporated to the ear fibroblasts of pig.Ganciclovir(GCV)and hygromycin(Hyg)were added into the culture medium for screening cells,and then cultured the cell to single cell clone.The integration of hCD55 was detected by PCR with two pairs of primers across the 5 'or 3' homology arms,respectively.The expression of hCD55 in the cells was detected by RT-PCR and Western blot.Ninety-four clones were obtained after the drug screening.Two clones were confirmed that hCD55 site-directed integration by PCR.The site-specific integration efficiency was 2.1%.The results of RT-PCR showed that the positive clones expressed hCD55 gene,and Western blot results further confirmed that hCD55 was expressed in the cells.This study successfully constructed a cell line expressing hCD55 on the basis of GTKO Bama minipig's ear fibroblast line,which would provide data foundation for the generation of xenotransplanted donor pigs.Conclusion: In this study,GTKO/hCD46/hCD55 expressing human dual complement regulatory protein xenograft donor pigs were successfully generated,using the GTKO/hCD46 Bama Wuzhishan hybrid porcine ear fibroblast cell line;GTKO Bama miniature pig ear fiber was successfully generated,based on the cell lines,a cell line that integrates and expresses the target gene hCD55 has been successfully constructed.The stability of the cloned experimental platform was verified and provided the basis for the preparation of xenotransplanted donor pigs.