赵平 刘佳 张颖.利用CRISPR/Cas9技术构建GLUT4基因敲减的A549细胞系[J].中南民族大学学报自然科学版,2020,39(2):133-138
利用CRISPR/Cas9技术构建GLUT4基因敲减的A549细胞系
Construction of GLUT4 knockdown A549 cell line based on CRISPR/Cas9 technologies
  
DOI:10.12130/znmdzk.20200205
中文关键词: CRISPR/Cas9  GLUT4  葡萄糖摄取
英文关键词: CRISPR/Cas9  GLUT4  glucose uptake
基金项目:国家自然科学基金资助项目(31070744);中央高校基本科研业务费专项基金资助项目(CZR18003);武汉市应用基础研究计划项目(2017060201010217);武陵山区特色资源植物物种保护与利用湖北省重点实验室项目(2018BFC360)
作者单位
赵平 刘佳 张颖 中南民族大学 生命科学学院医学生物研究所, 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&湖北省医学生物国际科技合作基地武汉 430074 
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中文摘要:
      目的:为探究GLUT4基因在肺癌细胞中的功能及影响.方法:在GLUT4外显子上设计了4个sgRNAs(S1、S2、S3、S4),分别与PX458载体连接形成重组表达载体后,借助转染试剂lipo3000将表达载体转入生长态势良好的A549细胞中,再利用流式细胞仪分选技术对转入重组载体发出绿色荧光的细胞进行单细胞分选.利用基因组测序筛选突变株,提取突变株的RNA和蛋白,进行Real-time PCR及Western Blot检测敲减效率,并利用激光共聚焦显微术检测突变株葡萄糖摄取量的变化.结果:突变株细胞在mRNA及蛋白水平均表现出基因敲减效果,并在葡萄糖摄取上表现出明显的降低趋势.结论:利用CRISPR/Cas9系统成功建立了GLUT4基因敲减A549细胞系.
英文摘要:
      Objective:To explore the function and effect of GLUT4 gene in lung cancer cells. Method:Four sgRNAs (S1, S2, S3 and S4) were designed on GLUT4 exon, which were connected with PX458 vector to form recombinant expression vectors. The expression vectors were then transfected into A549 cells with good growth status by transfection reagent lipo3000, and flow cytometry was used for sorting single cell. The mutants were screened by genome sequencing, and real-time PCR and Western Blot were used to detect the knockdown efficiency. Laser confocal microscopy was used to detect the change of glucose uptake in mutant strains. Results: The mutant cells showed gene knockdown effect at both the level of mRNA and protein, and was found significant decrease trend at glucose uptake. Conclusion: A GLUT4 gene knockdown A549 cell line was successfully established using CRISPR/Cas9 system.
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