利用CRISPR/Cas9编辑系统构建pAPN基因敲除的IPI-2I细胞系  

Establishment of pAPN Gene Knockout IPI-2I Cell Lines Mediated by CRISPR/Cas9 System

作　　者：徐长江 王晓朋 徐奎[1] 张秀玲[1] 向光明 赵海全 牟玉莲[1] 林晓 李奎[1] XU Changjiang;WANG Xiaopeng;XU Kui;ZHANG Xiuling;XIANG Guangming;ZHAO Haiquan;MU Yulian;LIN Xiao;LI Kui(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding,School of Life Science and Engineering,Foshan University,Foshan 528000,China;Nanning Strong Boar Bio-Technology Co.,Ltd.,Nanning 530001,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]佛山科学技术学院生命科学与工程学院,广东省动物分子设计与精准育种重点实验室,佛山528000 [3]南宁壮博生物科技有限公司,南宁530001

出　　处：《中国畜牧兽医》2021年第7期2282-2290,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31572378);转基因生物新品种培育重大专项(2018ZX08010-08B、2016ZX08010-004);中国农业科学院科技创新工程(ASTIP-IAS05)。

摘　　要：试验旨在采用CRISPR/Cas9技术获得猪氨基肽酶N(porcine aminopeptidase N,pAPN)基因敲除的猪回肠上皮(immortal pig intestinal-2I,IPI-2I)细胞系,进而在细胞水平上研究pAPN在冠状病毒入侵过程中的作用及相互作用机制。本研究将已构建好的靶向pAPN基因第2外显子上的2个sgRNA载体(pX330-GFP-g3和pX330-RFP-g5)共转染IPI-2I细胞。转染48 h后,荧光显微镜下观察IPI-2I细胞绿色荧光蛋白(green fluorescent protein,GFP)和红色荧光蛋白(red fluorescent protein,RFP)的发光情况,并用流式细胞仪收集带有GFP和RFP荧光标记的细胞,用来筛选单克隆细胞。然后取少量单克隆细胞提取DNA,用PCR扩增打靶区域附近的序列,测序鉴定其基因型,以获得pAPN基因敲除的IPI-2I细胞株。选择野生型和pAPN基因纯合片段敲除的IPI-2I细胞,提取细胞总蛋白,通过Western blotting检测pAPN基因敲除前后IPI-2I细胞中pAPN蛋白的表达。结果表明,转染48 h后,通过荧光显微镜可以观察到IPI-2I细胞中大部分细胞能够同时表达GFP和RFP,说明pX330-GFP-g3和pX330-RFP-g5质粒载体已经转入IPI-2I细胞。PCR和测序结果表明,共获得48株片段敲除单克隆细胞(片段敲除效率为15.5%),其中16株为单等位基因敲除,32株为双等位基因敲除;在32株双等位基因敲除细胞中,有23株细胞为纯合片段敲除。Western blotting结果表明,pAPN基因纯合片段敲除的细胞中检测不到pAPN蛋白的表达。综上,本研究利用CRISPR/Cas9编辑系统成功构建了pAPN基因纯合敲除的IPI-2I细胞系,为阐明pAPN在冠状病毒入侵过程中的作用机制以及制备猪抗病新品种奠定了基础。The purpose of this study was to establish porcine aminopeptidase N(pAPN)gene knockout IPI-2I cell lines by CRISPR/Cas9 system,and to further investigate the role and interaction mechanism of pAPN in the process of coronavirus invasion at the cellular level.Two sgRNA vectors(pX330-GFP-g3 and pX330-RFP-g5)targeting the second exon of the pAPN gene were co-transfected into IPI-2I cells.After 48 h of transfection,the GFP and RFP fluorescent was observed in most cells,and the positive cells with the RFP and GFP fluorescent label were sorted by flow cytometry for screening monoclonal cell line.Then genomic DNA was extracted from a small number of monoclonal cells,and the editing site of pAPN gene in monoclonal cell line was identified by PCR and sequenced for obtaining the pAPN gene knockout cell line.Before and after the pAPN gene knockout,the expression of pAPN protein in the IPI-2I cells was also detected by Western blotting.The results showed that most of the IPI-2I cells could simultaneously express GFP and RFP after 48 h of transfection,indicating that pX330-GFP-g3 and pX330-RFP-g5 vectors were transferred into IPI-2I cells.PCR and sequencing results suggested that a total of 48 fragment deletion monoclonal cells were obtained(the knockout efficiency was 15.5%),of which 16 were monoallelic knockout and 32 were biallelic knockout.Among 32 strains of biallelic knockout cells,23 strains of cells were homozygous fragment knockout.Western blotting results revealed that the expression of pAPN protein could not be detected in the homozygous fragment knockout cells.In summary,this study successfully constructed a pAPN gene homozygous fragment knockout IPI-2I cell line using CRISPR/Cas9 system,which laid the foundation for elucidating the mechanism of pAPN in the process of coronavirus invasion and preparing new pig breeds that could be resistant to disease.

关 键 词：猪氨基肽酶N CRISPR/Cas9 IPI-2I细胞 

分 类 号：S828.9[农业科学—畜牧学]