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.
[1] Campolo A, Laat M D, Keith L, et al.2016. Prolonged hyperinsulinemia affects metabolic signal transduction markers in a tissue specific manner[J]. Domestic Animal Endocrinology, 55: 41-45. [2] De Laat M A, McGowan C M, Sillence M N, et al.2010. Equine laminitis: Induced by 48 h hyperinsulinaemia in standardbred horses[J]. Equine Veterinary Journal, 42(2): 129-135. [3] Forbes B E, Blyth A J, Wit J M.2020. Disorders of IGFs and IGF-1R signaling pathways[J]. Molecular and Cellular Endocrinology, 518: 111035. [4] Fu Y, Foden J A, Khayter C, et al.2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J]. Nature Biotechnology, 31(9): 822-826 [5] Gibson D G, Young L, Chuang R Y, et al.2009. Enzymatic assembly of DNA molecules up to several hundred kilobases[J]. 6(5): 343-345. [6] Jiang W D, Bikard D, Cox D, et al.2013. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 31(3): 233-239. [7] Janik E, Niemcewicz M, Ceremuga M, et al.2020. Various aspects of a gene editing system-CRISPR-Cas9[J]. International Journal of Molecular Sciences, 21(24): 9604. [8] Knoll M S, Macher-Goeppinger S, Kopitz J, et al.2016. The ribosomal protein S6 in renal cell carcinoma: Functional relevance and potential as biomarker[J]. Oncotarget, 7(1): 418-432. [9] Kullmann A, Weber P S, Bishop J B, et al.2016. Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues[J]. Equine Veterinary Journal, 48(5): 626-632. [10] Lane H E, Burns T A, Hegedus O C, et al.2017. Lamellar events related to insulin-like growth factor-1 receptor signalling in two models relevant to endocrinopathic laminitis[J]. Equine Veterinary Journal, 49(5): 643-654. [11] Li G, Xing W, Min Z, et al.2018. Antifibrotic cardioprotection of berberine via downregulating myocardial IGF-1 receptor-regulated MMP-2/MMP-9 expression in diabetic rats[J]. American Journal of Physiology-Heart and Circulatory Physiology, 315(4): H802-H813. [12] Loftus J P, Johnson P J, Belknap J K, et al.2009. Leukocyte-derived and endogenous matrix metalloproteinases in the lamellae of horses with naturally acquired and experimentally induced laminitis[J]. Veterinary Immunology and Immunopathology, 129(3-4): 221-230. [13] Lorenc V E, Jaldin-Fincati J R, Luna J D, et al.2015. IGF-1 regulates the extracellular level of active MMP-2 and promotes muller glial cell motility[J]. Investigative Ophthalmology and Visual Science, 56(11): 6948-6960. [14] Memi F, Ntokou A, Papangeli I.2018. CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[J]. Seminars in Perinatology, 42(8): 487-500. [15] Nanayakkara S N, Rahnama S, Harris P A, et al.2019. Characterization of insulin and IGF-1 receptor binding in equine liver and lamellar tissue: Implications for endocrinopathic laminitis[J]. Domesticb Animal Endocrinology, 66: 21-26. [16] Pollak M.2008. Insulin and insulin-like growth factor signalling in neoplasia[J]. Nature Reviews Cancer, 8(12): 915-928. [17] Ran F A, Hsu P D, Wright J, et al.2013. Genome engineering using the CRISPR-Cas9 system[J]. Nature Protocols, 8(11): 2281-2308. [18] Rahnama S, Spence R, Vathsangam N, et al.2021. Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro[J]. Domesticb Animal Endocrinology, 74: 106530. [19] Riedemann J, Macaulay V M.2006. IGF1R signalling and its inhibition[J]. Endocrine-related Cancer, 13(Suppl 1): S33-43. [20] Zeng Z, Ren X, Yang S.2016. New methods for cloning large gene clusters based on CRISPR/cas9[J]. Chinese Journal of Biotechnology, 32(4): 401-408. [21] Zhang X H, Tee L Y, Wang X G, et al.2015. Off-target effects in CRISPR/Cas9-mediated genome engineering[J]. Molecular Therapy-Nucleic Acid, 4(11): e264. [22] Zhang Y, Gao C, Cao F, et al.2021. Pan-cancer analysis of IGF-1 and IGF-1r as potential prognostic biomarkers and immunotherapy targets[J]. Frontiers in Oncology,11: 755341.