CRISPR/Cas9介导敲除小鼠胚胎成纤维细胞IGF-1R基因的方法建立与验证

S823%S857.14+4%Q78; 胰岛素样因子1型受体(insulin-like growth factor 1 receptor,IGF-1R)基因在哺乳动物的生长发育和代谢中发挥着重要作用.本研究通过Gibson无缝链接(Gibson assembly)和重叠延伸PCR(genes plicing by overlap extension PCR,SOE PCR),利用pSMART LCKan载体将向导RNA(small guide RNA,sgRNA)插入lentiCRISPR v2质粒进行改造,构建IGF-1R基因敲除载体,通过菌液PCR、双酶切、基因测序对敲除载体进行评估;将该...

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Published in农业生物技术学报 Vol. 31; no. 2; pp. 416 - 424
Main Authors 于志勇, 刘萌萌, 姚枭洋, 胡学远, 接金磊, 迟良, 刘焕奇
Format Journal Article
LanguageChinese
Published 青岛农业大学巴瑟斯未来农业学院,青岛266109 2023
青岛农业大学动物医学院,青岛266109%青岛市智慧乡村发展服务中心,青岛266100%青岛农业大学动物医学院,青岛266109
Subjects
CRISPR/Cas9
小鼠胚胎成纤维细胞(MEF)
胰岛素样生长因子-1受体(IGF-1R)
载体构建
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Abstract S823%S857.14+4%Q78; 胰岛素样因子1型受体(insulin-like growth factor 1 receptor,IGF-1R)基因在哺乳动物的生长发育和代谢中发挥着重要作用.本研究通过Gibson无缝链接(Gibson assembly)和重叠延伸PCR(genes plicing by overlap extension PCR,SOE PCR),利用pSMART LCKan载体将向导RNA(small guide RNA,sgRNA)插入lentiCRISPR v2质粒进行改造,构建IGF-1R基因敲除载体,通过菌液PCR、双酶切、基因测序对敲除载体进行评估;将该载体转染至小鼠(Mus musculus)胚胎成纤维细胞中,通过Western blot比较敲除组和对照组细胞IGF-1R蛋白表达量.结果表明,IGF-1R基因敲除载体构建成功;敲除组细胞IGF-1R蛋白表达量极显著降低(P<0.01),表明成功敲除了小鼠胚胎成纤维细胞中IGF-1R基因.本研究改进了CRISPR/Cas9敲除系统表达载体的构建过程,为进一步在细胞层次研究IGF-1R基因的作用机制提供有力工具.
AbstractList S823%S857.14+4%Q78; 胰岛素样因子1型受体(insulin-like growth factor 1 receptor,IGF-1R)基因在哺乳动物的生长发育和代谢中发挥着重要作用.本研究通过Gibson无缝链接(Gibson assembly)和重叠延伸PCR(genes plicing by overlap extension PCR,SOE PCR),利用pSMART LCKan载体将向导RNA(small guide RNA,sgRNA)插入lentiCRISPR v2质粒进行改造,构建IGF-1R基因敲除载体,通过菌液PCR、双酶切、基因测序对敲除载体进行评估;将该载体转染至小鼠(Mus musculus)胚胎成纤维细胞中,通过Western blot比较敲除组和对照组细胞IGF-1R蛋白表达量.结果表明,IGF-1R基因敲除载体构建成功;敲除组细胞IGF-1R蛋白表达量极显著降低(P<0.01),表明成功敲除了小鼠胚胎成纤维细胞中IGF-1R基因.本研究改进了CRISPR/Cas9敲除系统表达载体的构建过程,为进一步在细胞层次研究IGF-1R基因的作用机制提供有力工具.
Author 迟良
姚枭洋
接金磊
胡学远
刘焕奇
于志勇
刘萌萌
AuthorAffiliation 青岛农业大学动物医学院,青岛266109%青岛市智慧乡村发展服务中心,青岛266100%青岛农业大学动物医学院,青岛266109;青岛农业大学巴瑟斯未来农业学院,青岛266109
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HU Xue-Yuan
LIU Meng-Meng
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LIU Huan-Qi
JIE Jin-Lei
CHI Liang
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DocumentTitle_FL Construction and Verification of IGF-1R Gene Knockout System in Mouse (Mus musculus) Embryonic Fibroblasts Based on CRISPR/Cas9
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Keywords CRISPR/Cas9
小鼠胚胎成纤维细胞(MEF)
胰岛素样生长因子-1受体(IGF-1R)
载体构建
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青岛农业大学动物医学院,青岛266109%青岛市智慧乡村发展服务中心,青岛266100%青岛农业大学动物医学院,青岛266109
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Title CRISPR/Cas9介导敲除小鼠胚胎成纤维细胞IGF-1R基因的方法建立与验证
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