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

作　　者：高登科 马白荣 郭怡莹 刘薇 刘田[1,2] 靳亚平 江舟[3] 陈华涛[1,2] GAO Deng-ke;MA Bai-rong;GUO Yi-ying;LIU Wei;LIU Tian;JIN Ya-ping;JIANG Zhou;CHEN Hua-tao(College of Veterinary Medicine,Northwest A&F University,Yangling 712100;Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs,Northwest A&F University,Yangling 712100;NHC Key Laboratory of Chronobiology,Sichuan University,Chengdu 610000)

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]西北农林科技大学农业农村部动物生物技术重点实验室,杨凌712100 [3]四川大学国家卫生健康委员会时间生物学重点实验室,成都610000

出　　处：《生物技术通报》2024年第2期65-72,共8页Biotechnology Bulletin

基　　金：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)。

摘　　要：【目的】利用CRISPR/Cas9技术构建小鼠胚胎成纤维细胞(NIH3T3)Quaking基因敲除细胞株,并检测Quaking基因对NIH3T3细胞增殖能力的影响。【方法】首先,利用在线网站设计两条靶向作用于Quaking外显子的sgRNA,成功构建了两个分别靶向Quaking基因第1、第2外显子的CRISPR/Cas9重组慢病毒质粒。将构建的Quaking基因CRISPR/Cas9重组慢病毒载体和pcDNA3.1-Quaking过表达质粒共转染至HEK293T细胞中,通过Western blot实验检测Quaking蛋白的敲除效率。其次,将筛选得到的敲除效率高的重组慢病毒质粒(LentiCRISPRv2-sgRNA1)与辅助包装质粒共转染入HEK293T细胞进行慢病毒包装,慢病毒转导NIH3T3细胞后,利用嘌呤霉素筛选阳性单克隆细胞株。最后,通过Western blot及免疫荧光染色鉴定敲除效果。【结果】发现Quaking蛋白在该细胞株中不表达,并测序证实了发生片段敲除。CCK8检测发现,Quaking基因敲除显著抑制了NIH3T3细胞的增殖能力。【结论】本研究首次通过CRISPR/Cas9技术成功构建了小鼠胚胎成纤维细胞(NIH3T3)Quaking基因敲除细胞株,为后续研究Quaking基因在小鼠生理功能调节中的作用机制提供了体外模型基础。【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 

分 类 号：Q78[生物学—分子生物学]