文献类型: 中文期刊
作者: 张雪萍 1 ; 刘嘉仪 1 ; 王彦芳 1 ; 吴添文 1 ;
作者机构: 1.中国农业科学院北京畜牧兽医研究所
关键词: 猪;CRISPR/Cas9;ACTA1基因;猪胎儿成纤维细胞(PEFs)
期刊名称: 中国畜牧兽医
ISSN: 1671-7236
年卷期: 2024 年 006 期
页码: 2273-2284
收录情况: 北大核心 ; CSCD
摘要: [目的]利用CRISPR/Cas9技术获得α-肌动蛋白1(actin alpha 1,ACTA1)基因敲除的猪胎儿成纤维细胞(porcine embryo fibroblasts, PEFs),为后续在细胞水平上探究猪ACTA1基因功能及发掘友好基因座位奠定基础.[方法]通过实时荧光定量PCR检测ACTA1基因在巴马小型猪心脏、肝脏、脾脏、肺脏、肾脏、背部皮下脂肪和背最长肌7种组织中的表达情况;利用CRISPOR在线网站在猪ACTA1基因第7外显子区域设计3条向导RNA(single guide RNA,sgRNA),并将其连接至pX330载体;通过T7E1酶切检测不同sgRNA活性,选择效率较高且符合目标的载体质粒共转染至PEFs中;利用有限稀释法筛选单克隆细胞,并对获得的单克隆细胞进行基因型鉴定和脱靶分析.[结果]实时荧光定量PCR结果表明,ACTA1基因在背最长肌中的表达量极显著高于其他组织(P<0.01).设计的3条sgRNA的基因编辑效率分别为24.87%(sgRNA1)、39.59%(sgRNA2)、36.93%(sgRNA3),综合切割位置和基因编辑效率,选用sgRNA1和sgRNA2共转染细胞.基因型鉴定结果表明,获得的69株单克隆细胞中有20株单克隆细胞发生了编辑,其中单等位基因片段敲除细胞3株,双等位基因片段敲除细胞1株,片段敲除效率为5.8%(4/69).脱靶分析结果显示,在预测的脱靶位点未检测到脱靶效应.[结论]本研究利用CRISPR/Cas9基因编辑系统成功构建了ACTA1基因纯合敲除的PEFs,研究结果为进一步探究ACTA1基因对猪骨骼肌发育调控提供了技术支撑和理论基础,同时为挖掘猪组织特异性表达的友好位点提供新思路.
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