薛璐, 欧阳聪, 孙宁远, 秦绪慧.基于CRISPR/Cas9技术建立敲除Plac9基因的人胚肾细胞株[J].中南民族大学学报自然科学版,2019,(2):189-192
基于CRISPR/Cas9技术建立敲除Plac9基因的人胚肾细胞株
Establishment of Plac9 Gene Knockout 293T Cell Line via CRISPR/Cas9 Technique
  
DOI:10.12130/znmdzk.20190207
中文关键词: CRISPR/Cas9  基因编辑技术  基因敲除  Plac9
英文关键词: CRISPR/Cas9  genome editing technique  gene knockout  Plac9
基金项目:国家自然科学基金资助项目 (31101047)
作者单位
薛璐, 欧阳聪, 孙宁远, 秦绪慧 中南民族大学 生命科学学院生物医学研究所武陵山区特色资源植物种质保护与利用湖北省重点实验室武汉430074 
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中文摘要:
      目的:通过CRISPR/Cas9基因编辑技术和流式细胞术相结合,获得可编辑Plac9基因敲除的人胚肾细胞株(293T).方法:根据Plac9外显子设计了4个sgRNAs(S1,S2,S3,S4),分别将其与PX458载体连接,构建了PX458-S1(S2/S3/S4)载体.通过转染试剂lipo2000将表达载体分别转染至293T细胞中,并以流式细胞术对带绿色荧光蛋白标记的细胞进行单细胞分选,分选后的细胞培养一段时间后,用基因组测序和错配酶酶切进行筛选.从筛选好的细胞中提取蛋白,进行Western Blot检测敲除效率.结果:采用CRISPR/Cas9和流式细胞术结合技术成功构建了Plac9蛋白表达缺失的人胚肾细胞株. 结论:该方法简便快捷、效率高,可广泛地用于编辑各种细胞和细胞功能研究.
英文摘要:
      Objective: To obtain editable Plac9 gene knockout 293T cell line through CRISPR/Cas9 gene editing and flow cytometry. Methods: 4 sgRNAs (S1,S2,S3,S4) were designed and respectively connected with PX458 carrier, according to the exons of Plac9 gene, then vectors PX458-S1(S2/S3/S4)were constructed. 293T cells were transfected with the expression vectors by transfection reagent lipo2000. The green fluorescent protein labeled cells were then concentrated via single cell sorting by flow cytometry. Selected cells were cultured and identified with genomic sequencing and mismatched enzyme digestion. The protein was extracted from the selected cells and the knockout efficiency was measured by Western Blot. Results: Protein Plac9 knockout 293T cell line was successfully constructed by employing CRISPR/Cas9 and flow cytometry technology. Conclusion: This method is convenient and efficient, which can be widely applied for editing any type of cell lines and for the study of cell functions.
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