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CRISPR/Cas9介导的CD71基因编辑IPI-2I细胞系的建立

文献类型: 中文期刊

作者: 聂雨欣 1 ; 袁茂莎 1 ; 王婉洁 1 ; 王楠 2 ; 孙亚茹 1 ; 刘志国 1 ; 牟玉莲 1 ;

作者机构: 1.中国农业科学院北京畜牧兽医研究所

2.中国农业科学院(深圳)农业基因组研究所

关键词: 猪;CRISPR/Cas9;CD71基因;IPI-2I细胞

期刊名称: 中国畜牧兽医

ISSN: 1671-7236

年卷期: 2025 年 52 卷 010 期

页码: 4527-4537

收录情况: 北大核心

摘要: [目的]通过CRISPR/Cas9技术获得分化抗原71(cluster of differentiation 71,CD71)基因编辑的猪回肠上皮(immortalized porcine intestinal-2I,IPI-2I)细胞系,为进一步在细胞水平上研究CD71基因功能及其在猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)感染中的作用提供材料.[方法]针对CD71基因第3外显子及其相邻内含子区域设计5条sgRNAs,并将其连接至pX458-GFP载体;在猪胎儿成纤维(porcine embryonic fibroblast,PEF)细胞中通过T7E1酶切试验对不同sgRNA载体进行活性检测;选择切割效率较高的2个sgRNAs载体质粒分别电转染至IPI-2I细胞中,48 h后通过流式细胞仪收集带有绿色荧光蛋白(green fluorescent protein,GFP)的单个细胞至96孔细胞培养板中扩大培养,筛选单克隆细胞;通过PCR扩增和TA克隆技术对单克隆细胞进行基因型鉴定,获得CD71基因编辑的IPI-2I细胞系.[结果]质粒测序结果表明,5条sgRNAs均成功连接至pX458-GFP载体;T7E1酶切结果显示,转染的5个sgRNAs质粒组均产生了切割,其中,pX458-sgRNA1和pX458-sgRNA4切割效率较高,分别为35.8%和44.1%.流式细胞术分选结果显示,转染pX458-sgRNA1和pX458-sgRNA4的阳性细胞比例分别为29.8%和25.7%;PCR扩增结果显示,获得的65株单克隆细胞中有14株细胞发生了基因编辑,编辑效率为22%;TA克隆结果显示,14株基因编辑细胞中有5株为单等位基因编辑,9株为双等位基因编辑.[结论]本研究利用CRISPR/Cas9技术成功构建了 CD71基因编辑的IPI-2I细胞系,获得了 CD71基因单等位基因编辑和双等位基因编辑细胞.试验结果为明确CD71基因功能及其介导TGEV感染的作用机制提供了良好的细胞模型.

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