GGTA1/β4GalNT2双基因敲除近交系五指山小型猪的建立
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摘要
目的:利用CRISPR/Cas9技术建立五指山小型猪近交系GGTA1/β4GalNT2双基因敲除克隆猪。方法:设计合成靶向猪GGTA1和β4GalNT2基因的单导向RNA(single guide RNA,sgRNA),以pX330质粒为骨架,分别构建GGTA1和β4GalNT2的Cas9打靶载体,转染至近交系五指山小型猪胎儿成纤维细胞(porcine fetal fibroblast,PFF)中,通过G418药物筛选和测序鉴定获得双基因敲除的单细胞克隆,然后利用体细胞克隆技术(somatic cell nuclear transfer,SCNT)获得双基因敲除的近交系五指山小型猪,并利用流式细胞技术检测克隆猪外周血单核细胞(peripheral blood mononuclear cell,PBMC)中αGal和Sd(a)抗原的表达。结果:成功构建GGTA1和β4GalNT2基因的Cas9/sgRNA表达载体,转染后获得双基因敲除的近交系五指山小型猪PFF细胞克隆9个。SCNT成功获得了10只五指山小型猪近交系双基因敲除的克隆猪,其PBMC无αGal和Sd(a)抗原的表达。结论:CRISPR/Cas9技术可以实现对猪GGTA1/β4GalNT2基因的编辑。本实验首次成功制备了GGTA1/β4GalNT2双基因敲除的近交系五指山型猪,为异种器官移植研究与应用提供了新的供体材料。
Objective:This study aims to build inbred Wuzhishan miniature pigs with GGTA1/β4 GalNT2 double gene knockout by CRISPR/Cas9 mediated targeting. Methods: Single-guide RNAs(sgRNAs)specific for the pig GGTA1 and β4 GalNT2 were designed and synthesized,then cloned into the pX330 plasmid containing a Cas9 skeleton,respectively. The resulting targeting vectors for GGTA1 and β4 GalNT2 were transfected into the primary porcine fetal fibroblasts(PFFs)derived from Wuzhishan miniature inbred pigs. G418 drug screening and Sanger sequencing were used to identify the monoclonal cells with GGTA1/β4 GalNT2 double gene knockout. Somatic cell nuclear transfer(SCNT)was employed to generate Wuzhishan miniature inbred pigs using the obtained colonies as donor cells. Flow cytometry analysis was conducted to examine the αGal and Sd(a)antigen expression in peripheral blood mononuclear cell(PBMC)of the cloned piglets. Results:Cas9/sgRNA expression vectors targeting pig GGTA1 and β4 GalNT2 genes were successfully constructed. After transfection into PFFs,9 cell colonies were obtained with biallelic modifications in both GGTA1 and β4 GalNT2 loci. Ten cloned piglets were produced by SCNT. The expression of αGal and Sd(a)antigens on these cloned knockout piglets was negative. Conclusion:The CRISPR/Cas9 system showed high efficiency in pig gene targeting. The GGTA1/β4 GalNT2 double gene knockout inbred Wuzhishan miniature pigs were successfully produced in the present study,which could serve as newly ideal materials for xenotransplantation.
Objective:This study aims to build inbred Wuzhishan miniature pigs with GGTA1/β4 GalNT2 double gene knockout by CRISPR/Cas9 mediated targeting. Methods: Single-guide RNAs(sgRNAs)specific for the pig GGTA1 and β4 GalNT2 were designed and synthesized,then cloned into the pX330 plasmid containing a Cas9 skeleton,respectively. The resulting targeting vectors for GGTA1 and β4 GalNT2 were transfected into the primary porcine fetal fibroblasts(PFFs)derived from Wuzhishan miniature inbred pigs. G418 drug screening and Sanger sequencing were used to identify the monoclonal cells with GGTA1/β4 GalNT2 double gene knockout. Somatic cell nuclear transfer(SCNT)was employed to generate Wuzhishan miniature inbred pigs using the obtained colonies as donor cells. Flow cytometry analysis was conducted to examine the αGal and Sd(a)antigen expression in peripheral blood mononuclear cell(PBMC)of the cloned piglets. Results:Cas9/sgRNA expression vectors targeting pig GGTA1 and β4 GalNT2 genes were successfully constructed. After transfection into PFFs,9 cell colonies were obtained with biallelic modifications in both GGTA1 and β4 GalNT2 loci. Ten cloned piglets were produced by SCNT. The expression of αGal and Sd(a)antigens on these cloned knockout piglets was negative. Conclusion:The CRISPR/Cas9 system showed high efficiency in pig gene targeting. The GGTA1/β4 GalNT2 double gene knockout inbred Wuzhishan miniature pigs were successfully produced in the present study,which could serve as newly ideal materials for xenotransplantation.
引文
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[11]宋宗培,郭蝶,蔡志明,等.异种器官移植免疫生物学研究进展[J].器官移植,2018,9(3):236-238
[12]Zhang RJ,Wang Y,Chen L,et al. Reducing immunoreactivity of porcine bioprosthetic heart valves by geneticallydeleting three major glycan antigens,GGTA1/β4GalNT2/CMAH[J]. Acta Biomater,2018,72:196-205
[2] Byrne GW,Du Z,Stalboerger P,et al. Cloning and expression of porcineβ1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen[J]. Xenotransplantation,2014,21(6):543-554
[3] Estrada JL,Martens G,Li P,et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes[J]. Xenotransplantation,2015,22(3):194-202
[4]王俊政,李艳如,赵丽华,等. CRISPR/Cas9介导的猪Six1和Six4基因敲除细胞系的建立[J].南京医科大学学报(自然科学版),2018,38(2):143-148
[5]曾华沙,姚俊,王红顺,等.人/猪OSBPL2同源性比较及猪PFFs靶基因敲除细胞系的建立[J].南京医科大学学报(自然科学版),2018,38(2):149-154
[6]尹智,袁雪薇,吕嘉伟,等. CRISPR/Cas9系统介导的一步法胚胎注射获得猪GGTA1敲除胚胎[J].畜牧与兽医,2016,48(4):15-18
[7]唐雨婷,高景波,龙川,等. CRISPR/Cas9介导的β4GalNT2基因敲除猪制备[J].农业生物技术学报,2017,25(10):1697-1705
[8]冯书堂,李奎,刘岚,等.小型猪近交系新品种的培育与开发利用[J].农业生物技术学报,2015,23(2):274-280
[9]张青,周翠冰,戴一凡,等.神经细胞异种移植的研究进展[J].器官移植,2017,8(4):328-332
[10]周明,邓阳阳,戴一凡,等.猪肺异种移植的研究进展与发展方向[J].器官移植,2017,8(6):476-479
[11]宋宗培,郭蝶,蔡志明,等.异种器官移植免疫生物学研究进展[J].器官移植,2018,9(3):236-238
[12]Zhang RJ,Wang Y,Chen L,et al. Reducing immunoreactivity of porcine bioprosthetic heart valves by geneticallydeleting three major glycan antigens,GGTA1/β4GalNT2/CMAH[J]. Acta Biomater,2018,72:196-205