【摘要】 人补体调节蛋白(human complement regulatory protein,h CRP)基因,如人类簇分化抗原55(human cluster of differentiation 55,hCD55),在异种器官移植中尤为重要。本研究在α-1,3半乳糖苷转移酶(α-1,3-galactosyltransferase,GGTA1)基因敲除(α-1,3-galactosyltransferase knockout,GTKO)巴马小型猪(Sus scrofa)耳成纤维细胞的基础上,利用CRISPR/Cas9技术在猪Rosa26(reverse orientation splice acceptorβ-geo26)基因座敲入hCD55基因,以制备hCD55基因高效表达的细胞系。分别构建靶向切割pRosa26位点intron 1(内含子1)的Cas9表达载体,及pRosa26位点同源重组hCD55的表达载体,后者包含异源Loxp位点间的EF1α(polypeptide chain elongation factor 1α)启动子调控的hCD55基因、潮霉素B磷酸转移酶基因(hygromycin B phosphotransferase,HPH)、胸腺激酶基因(thymidine kinase,TK)为正负筛选基因。将Cas9-Rosa26表达载体和p EF1α-hCD55表达载体共电转染猪耳成纤维细胞系,培养液中加入丙氧鸟苷(Ganciclovir,GCV)和潮霉素(hygromycin,Hyg)进行筛选,培养至单细胞克隆。通过跨5’端和3’端同源臂的两对引物PCR检测目的基因整合情况,反转录(reverse transcription PCR,RT-PCR)和Western blot检测hCD55在细胞中的表达情况。药物筛选后得到94个克隆点,经PCR鉴定,定点整合hCD55的阳性克隆点2个,定点整合效率为2.1%。RT-PCR结果显示,阳性克隆点细胞表达hCD55基因,Western blot结果进一步证实hCD55在细胞有表达。本研究在GTKO巴马小型猪耳成纤维细胞系基础上成功构建了表达目的基因hCD55的细胞系,为异种移植供体猪的制备提供了基础资料。更多还原

【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 Cas9 expression vector targeted the intron 1(intron 1) of the reverse orientation splice acceptor β-geo2(Rosaβgeo26) site in swine.A expression vector homologous-recombinating hCD55 at the pRosa26 site werconstructed,which contained the the hCD55 gene under the control of polypeptide chain elongation factor 1(EF1α) in the heterogeneous Loxps.The hygromycin B phosphotransferase(HPH) gene was the positivscreening gene.The thymidine kinase(TK) was the negative gene.The Cas9-Rosa26 expression vector anthe p EF1α-hCD55 expression vector were co-electroporated to the ear fibroblasts of pig.Ganciclovir(GCVand hygromycin(Hyg) were added into the culture medium for screening cells,and then cultured the cell tsingle 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 werconfirmed that hCD55 site-specific integration by PCR.The site-specific integration efficiency was 2.1%.Thresults of RT-PCR showed that the positive clones expressed hCD55 gene,and Western blot results furtheconfirmed that hCD55 was expressed in the cells.This study successfully constructed a cell line expressinhCD55 on the basis of GTKO Bama mini pig’s ear fibroblast line,which would provide data foundation for thgeneration of xenotransplanted donor pigs.更多还原