Abstract：Abstract Efficient expression of human complement regulatory gene human cluster of differentiation 55(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 (α-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 β-geo26 (Rosaβgeo26) 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 1α(EF1α) 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 pEF1α-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 reverse transcription PCR(RT-PCR) and Western blot. Ninety-four clones were obtained after the drug screening. Two clones were confirmed that hCD55 site-specific 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 mini pig's ear fibroblast line, which would provide data foundation for the generation of xenotransplanted donor pigs.
