Construction and Verification of IGF-1R Gene Knockout System in Mouse (Mus musculus) Embryonic Fibroblasts Based on CRISPR/Cas9
YU Zhi-Yong1, LIU Meng-Meng3, YAO Xiao-Yang1, HU Xue-Yuan1, JIE Jin-Lei1, CHI Liang1,*, LIU Huan-Qi1,2,*
1 College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China; 2 Bathurst Future Agri-Tech Institute, Qingdao Agricultural University, Qingdao 266109, China; 3 Qingdao Smart Village Development Service Center, Qingdao 266100, China
Abstract：Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in the growth, development and metabolism of mammals. In this study, the small guide RNA (sgRNA) was inserted into lentiCRISPR v2 plasmid by Gibson assembly and gene splicing by overlap extension PCR (SOE PCR), and pSMART LCKan vector was used to construct IGF-1R gene knockout vector. The vector was verified by bacterial liquid PCR, double enzyme digestion and gene sequencing. After that, the vector was verified by bacterial liquid PCR, double enzyme digestion. The expression of IGF-1R protein in knockout group and control group was compared by Western blot. The results of PCR, double enzyme digestion and gene sequencing showed that the vector was successfully constructed. Western blot showed that the expression of IGF-1R protein decreased extremely significantly in the knockout group (P<0.01), indicating the successful knockout of IGF-1R gene in mouse (Mus musculus) embryonic fibroblast (MEF) cells. This study improved the construction process of expression vector of CRISPR/Cas9 knockout system, and provides a powerful tool for further studying the role of IGF-1R gene at the cellular level.
于志勇, 刘萌萌, 姚枭洋, 胡学远, 接金磊, 迟良, 刘焕奇. 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[J]. 农业生物技术学报, 2023, 31(2): 416-424.
YU Zhi-Yong, LIU Meng-Meng, YAO Xiao-Yang, HU Xue-Yuan, JIE Jin-Lei, CHI Liang, LIU Huan-Qi. Construction and Verification of IGF-1R Gene Knockout System in Mouse (Mus musculus) Embryonic Fibroblasts Based on CRISPR/Cas9. 农业生物技术学报, 2023, 31(2): 416-424.
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