基础医学与临床 ›› 2018, Vol. 38 ›› Issue (5): 589-593.

• 研究论文 •    下一篇

利用CRISPR /CAS9技术构建QKI敲除GC1-spg细胞株及其生物学功能检测

钟顺顺1,李凯2,杨阳1,缪时英3,王琳芳3,宋伟4   

  1. 1. 中国医学科学院基础医学研究所
    2. 中国医学科学院
    3. 中国医学科学院 基础医学研究所
    4. 中国医学科学院基础医学研究所,北京协和医学院基础学院,医学分子生物学国家重点实验室,生物化学与分子生物学系
  • 收稿日期:2018-02-06 修回日期:2018-03-20 出版日期:2018-05-05 发布日期:2018-04-28
  • 通讯作者: 宋伟 E-mail:roy_sw0925@yahoo.com.cn
  • 基金资助:
    国家重大科学计划

Construction and characterization of QKI gene knockout GC1-spg cell strain with CRISPR/CAS9

  • Received:2018-02-06 Revised:2018-03-20 Online:2018-05-05 Published:2018-04-28
  • Contact: Wei SONG E-mail:roy_sw0925@yahoo.com.cn

摘要: 目的 利用CRISPR/Cas9 技术构建QKI 基因敲除的GC1-spg细胞系,并在体外研究QKI对GC1-spg细胞增殖和分化的影响。方法 利用CRISPR/Cas9系统的PX330质粒构建敲除QKI基因的重组质粒,转染GC1-spg野生型细胞,加入嘌呤霉素进行筛选,通过蛋白质免疫印迹(Western Blot)和基因测序鉴定出GC1-spg敲除QKI基因细胞系;正常培养野生型和敲除细胞系,利用细胞计数试剂盒(CCK8)检测增殖曲线和荧光定量PCR(q-PCR)检测减数分裂相关基因变化,从而研究QKI蛋白在GC1-spg增殖与分化中的重要作用。结果 本研究成功构建出QKI基因敲除GC1-spg细胞系,与对照组相比,QKI敲除细胞系的增殖明显下降(*P < 0.05),减数分裂相关分子标志基因表达量明显降低(*P < 0.05)。结论 QKI蛋白可以影响GC1-spg的增殖和分化,因此QKI蛋白可能通过影响增殖和分化而影响精子发生。

关键词: CRISPR/CAS9, GC1-spg, QKI, 精子发生

Abstract: Objective To investigate whether QKI protein plays an important role in the process of spermatogenesis. Constructing GC1-spg cell line which knocked out QKI gene by the technology of CRISPR / Cas9 ,and detecting its effect on the proliferation and differentiation of QKI protein in vitro. Methods The plasmid PX330 was used to construct QKI gene knockout recombinant plasmid, then was transfected it to GC1-spg wild-type cells and was selected it by puromycin, and GC1-spg knock-QKI gene cell lines were identified by Western Blot and gene sequencing; The wild-type and knockout cell lines were cultured normally, then detected the growth curve by cell counting kit (CCK8) , and using quantitative PCR to get the changes of meiotic-related gene differentiation. Results In this study, QKI gene knockout GC1-spg cell line was successfully constructed. Compared with the control group, the growth of QKI knockout cell line was significantly decreased (*P < 0.05), and the expression of meiosis related molecular marker gene was significantly decreased (*P < 0.05). Conclusions QKI proteins can affect reproductive spermatogenesis by acting on proliferation and differentiation.

Key words: CRISPR/CAS9, GC1-spg, QKI, Spermatogenesis

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