利用CRISPR/Cas9技术构建Quaking敲除的小鼠胚胎成纤维细胞株

doi:10.13560/j.cnki.biotech.bull.1985.2023-0879

生物技术通报 ›› 2024, Vol. 40 ›› Issue (2): 65-72.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

高登科¹^,²(), 马白荣¹^,², 郭怡莹¹, 刘薇¹^,², 刘田¹^,², 靳亚平¹^,², 江舟³(), 陈华涛¹^,²()

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

收稿日期:2023-09-12 出版日期:2024-02-26 发布日期:2024-03-13
通讯作者: 陈华涛，男，博士，教授，研究方向：哺乳动物生物钟调控生殖与代谢机制；E-mail: htchen@nwafu.edu.cn；
江舟，男，博士，副研究员，研究方向：时间生物学；E-mail: jweiz@126.com
作者简介:高登科，男，博士研究生，研究方向：哺乳动物生物钟调控代谢机制；E-mail: gdk960101@nwafu.edu.cn
第一联系人：马白荣为共同第一作者
基金资助:
国家自然科学基金面上项目(32373088);国家自然科学基金面上项目(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)

Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology

GAO Deng-ke¹^,²(), MA Bai-rong¹^,², GUO Yi-ying¹, LIU Wei¹^,², LIU Tian¹^,², JIN Ya-ping¹^,², JIANG Zhou³(), CHEN Hua-tao¹^,²()

1. College of Veterinary Medicine, Northwest A&F University, Yangling 712100
2. Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling 712100
3. NHC Key Laboratory of Chronobiology, Sichuan University, Chengdu 610000

Received:2023-09-12 Published:2024-02-26 Online:2024-03-13

摘要/Abstract

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。

关键词: Quaking, CRISPR/Cas9, 小鼠胚胎成纤维细胞, 基因敲除

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.

Key words: Quaking, CRISPR/Cas9, mouse embryonic fibroblasts, gene knockout

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.

GAO Deng-ke, MA Bai-rong, GUO Yi-ying, LIU Wei, LIU Tian, JIN Ya-ping, JIANG Zhou, CHEN Hua-tao. Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2024, 40(2): 65-72.

图/表 9

表1 序列信息

Table 1 Sequences information

名称Name	序列Sequence（5'-3'）
sgRNA1	CACCGAAGTGCAGAATTGCCTGACG
sgRNA1	AAACCGTCAGGCAATTCTGCACTTC
sgRNA2	CACCGGATCTTCAACCACCTCGAG
sgRNA2	AAACCTCGAGGTGGTTGAAGATCC
Target-F	TTATTTCCCAAAGTGAC
Target-R	ATGCCCGAATAGGTT

图1 小鼠Quaking基因的sgRNA设计图

Fig. 1 sgRNA design of Quaking gene in mice

图2 重组质粒的构建与鉴定结果 A：LentiCRISPRv2空载体酶切电泳图，M: marker；1: Esp3I酶切后的LentiCRISPRv2空载体；B：重组质粒LentiCRISPRv2-sgRNA1和LentiCRISPRv2-sgRNA2的测序结果

Fig. 2 Construction and identification of recombinant plasmids A: Electrophoresis of LentiCRISPRv2 empty vector enzyme digestion, M: marker; 1: empty carrier of LentiCRISPRv2 after digestion by Esp3I. B: Sequencing results of recombinant plasmids LentiCRISPRv2-sgRNA1 and LentiCRISPRv2-sgRNA2

图3 Western blot筛选QKI蛋白敲除效率高的重组质粒 1：未转染的空白细胞对照；2：转染pcDNA3.1-Quaking过表达载体；3：共转染pcDNA3.1-Quaking和LentiCRISPRv2空载体；4：共转染pcDNA3.1-Quaking和LentiCRISPRv2-sgRNA1；5：共转染pcDNA3.1-Quaking和LentiCRISPRv2-sgRNA2

Fig. 3 Screening of recombinant plasmids with high QKI protein knockout efficiency via Western blot 1: Blank control cells without transfection; 2: transfected pcDNA3.1-Quaking overexpression vector; 3: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2 empty vectors; 4: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2-sgRNA1; 5: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2-sgRNA2

图4 Quaking敲除细胞株与野生型细胞株测序结果比对示意图 Wild-type：Quaking野生型细胞株序列；Quaking KO：Quaking敲除细胞株序列；sgRNA用黑色标记，PAM序列用红色标记，蓝色虚线表示删除碱基，括号中的数字表示删除碱基数

Fig. 4 Schematic diagram of comparison between sequencing results of Quaking knockout cell lines and wild-type cell lines Wild-type: Quaking wild-type cell line sequence; Quaking KO: Quaking knockout cell line sequence; sgRNAs are marked in black, PAM sequences in red, dashed blue lines indicate the number of bases removed, and numbers in parentheses indicate the number of bases removed

图5 NIH3T3细胞形态图 A：对照组NIH3T3细胞形态；B：Quaking基因敲除的NIH3T3形态

Fig. 5 Morphology of NIH3T3 cells A: NIH3T3 cell morphology in control group; B: Quaking gene knockout NIH3T3 morphology

图6 Western blot检测QKI蛋白表达 1：对照组NIH3T3细胞样品；2：Quaking基因敲除的NIH3T3细胞样品

Fig. 6 Detection of QKI protein expression via Western blot 1: NIH3T3 cell samples in control group; 2: NIH3T3 cell samples knocked out by Quaking gene

图7 免疫荧光染色检测QKI蛋白表达

Fig. 7 QKI protein expression detected by immunofluorescence staining

图8 Quaking基因敲除对NIH3T3细胞增殖能力的影响 P < 0.001表示双因素方差分析两组间细胞增殖活力差异极显著；***P < 0.001表示差异极显著

Fig. 8 Effects of Quaking gene knockout on the proliferation ability of NIH3T3 cells P < 0.001 indicates that the cell proliferation activity was significantly different between the two groups in two-factor ANOVA; and ***P < 0.001 indicates a very significant difference

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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

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