生物技术通报2024，Vol.40Issue(2)：65-72,8.DOI:10.13560/j.cnki.biotech.bull.1985.2023-0879
利用CRISPR/Cas9技术构建Quaking敲除的小鼠胚胎成纤维细胞株
Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology
摘要
Abstract
[Objective]CRISPR/Cas9 technology was used to generate a mouse embryonic fibroblast cell line(NIH3T3)with a knockout of the Quaking gene and to investigate its impact on NIH3T3 cell proliferation.[Method]Initially,two sgRNAs targeting Quaking exons were designed using an online platform,and two CRISPR/Cas9 recombinant lentiviral plasmids targeting the first and second exons of the Quaking gene were constructed successfully.These constructs with pcDNA3.1-Quaking overexpression plasmids were co-transfected into HEK293T cells,and the knockout efficiency of Quaking protein was assessed through Western blot analysis.Subsequently,the recombinant lentiviral plasmid(LentiCRISPRv2-sgRNA1)with high knockout efficiency was co-transfected with auxiliary packaging plasmids into HEK293T cells for lentivirus packaging.After lentiviral transduction of NIH3T3 cells,positive monoclonal cell lines were selected using puromycin.Finally,we confirmed the knockout effect through Western blot and immunofluorescence staining,demonstrating the absence of Quaking protein in these cells.[Result]Sequencing confirmed the occurrence of a targeted gene segment deletion.CCK8 assays revealed that Quaking gene knockout significantly inhibited NIH3T3 cell proliferation.[Conclusion]This study represents the first successful utilization of CRISPR/Cas9 technology to establish a Quaking gene knockout cell line in mouse embryonic fibroblast cells(NIH3T3),providing a valuable in vitro model for exploring the mechanistic role of the Quaking gene in the regulation of mouse physiological functions.关键词
Quaking/CRISPR/Cas9/小鼠胚胎成纤维细胞/基因敲除Key words
Quaking/CRISPR/Cas9/mouse embryonic fibroblasts/gene knockout引用本文复制引用
高登科,马白荣,郭怡莹,刘薇,刘田,靳亚平,江舟,陈华涛..利用CRISPR/Cas9技术构建Quaking敲除的小鼠胚胎成纤维细胞株[J].生物技术通报,2024,40(2):65-72,8.基金项目
国家自然科学基金面上项目(32373088,31771301),国家卫生健康委员会时间生物学重点实验室(四川大学)开放基金资助项目(NHCC-2022-01),中国食品科学技术学会食品科技基金-雅培食品营养与安全专项科研基金(2022-F06) （32373088,31771301）
