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

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

目的利用CRISPR/Cas9基因编辑技术构建QKI敲除的GC1-spg细胞株,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细胞株;常规培养野生型和敲除细胞株,利用细胞计数检测试剂盒(CCK8)绘制增殖曲线和荧光定量PCR(q-PCR)检测减数分裂相关基因变化。结果本研究成功构建出QKI敲除GC1-spg细胞株,与对照组相比,QKI敲除细胞株的增殖明显下降(P<0.05),减数分裂相关分子标志基因c-kit、Mtl5和Hspa2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。

Objective To investigate whether QKI protein plays any important role in the process of spermatogenesis. Constructing GC1-spg cell strain which knocked out QKI 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 knockout recombinant plasmid,then transfected it to GC1-spg wild-type cells and selected by puromycin. GC1-spg knock-QKI cell strain was identified by Western blot and gene sequencing; The wild-type and knockout cell strain was 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 QKI knockout GC1-spg cell strain was successfully constructed. Compared with the control group,the growth of QKI knockout cell strain was significantly decreased（ P〈0. 05）,and the expression of meiosis related molecular marker gene of c-kit,Mtl5 and Pspa2 was significantly decreased（ P〈0. 05）. Conclusions QKI proteins can affect reproductive spermatogenesis by acting on proliferation and differentiation.