CAJ | 학술논문

目的：利用改良的CRISPR/Cas9基因编辑系统敲除HeLa细胞中SND1基因，构建HeLa细胞SND1基因敲除稳定株。方法：设计一对特异性识别SND1基因第二个启动子的上下游sgRNA，以PX462质粒为载体，构建出一对重组真核表达质粒。酶切和测序鉴定后，将一对重组质粒共同转染进入HeLa细胞中，使用嘌呤霉素进行阳性细胞筛选，挑取单克隆细胞进行培养。最后用Western Blot鉴定敲除效果。结果：sgRNA正确插入到PX462质粒载体中，转染并筛选单克隆后的细胞中没有SND1蛋白的表达。结论：成功构建出HeLa细胞SND1基因敲除稳定株。
목적：이용개량적CRISPR/Cas9기인편집계통고제HeLa세포중SND1기인，구건HeLa세포SND1기인고제은정주。방법：설계일대특이성식별SND1기인제이개계동자적상하유sgRNA，이PX462질립위재체，구건출일대중조진핵표체질립。매절화측서감정후，장일대중조질립공동전염진입HeLa세포중，사용표령매소진행양성세포사선，도취단극륭세포진행배양。최후용Western Blot감정고제효과。결과：sgRNA정학삽입도PX462질립재체중，전염병사선단극륭후적세포중몰유SND1단백적표체。결론：성공구건출HeLa세포SND1기인고제은정주。
Objective:To apply modified CRISPR/Cas9 gene editing system to knock out the SND1 gene in HeLa cell and construct HeLa SND1 gene knockout stable strain. Methods:A pair of sgRNAs that could specially identify the upstream and downstream of SND1 gene second promoter were designed, then a recombinant eukaryotic expressional plasmid by the carrier of PX462 was constructed. After enzyme digestion and sequencing, a pair of recombinant plasmids into HeLa cell were co-transfected, then puromycin was used to screen positive cell and the monoclonal cell was developed. The knockout effect was measured by western blotting. Results:sgRNA was correctly inserted into the PX462 recombinant plasmid, and SND1 protein was undetected in HeLa cell after transfection and screening of monoclonal cell. Conclusion:HeLa SND1 gene knockout stable strain can be successfully built.