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应用CRISPR/Cas9技术构建IL-15敲除的MGC-803胃癌细胞株
引用本文:俞 静,孙 丽,孙晓仙,陈志红,钱 晖,许文荣,朱 伟.应用CRISPR/Cas9技术构建IL-15敲除的MGC-803胃癌细胞株[J].南京医科大学学报,2016(2):140-143.
作者姓名:俞 静  孙 丽  孙晓仙  陈志红  钱 晖  许文荣  朱 伟
作者单位:江苏大学医学院检验系,江苏 镇江 212013,江苏大学医学院检验系,江苏 镇江 212013,江苏大学医学院检验系,江苏 镇江 212013,江苏大学附属人民医院外科,江苏 镇江 212001,江苏大学医学院检验系,江苏 镇江 212013,江苏大学医学院检验系,江苏 镇江 212013;江苏大学附属医院检验科,江苏 镇江 212001,江苏大学医学院检验系,江苏 镇江 212013
基金项目:国家自然科学基金资助(81472334,81270214)
摘    要:目的:运用CRISPR/Cas9基因编辑技术,建立IL-15基因敲除的MGC-803胃癌细胞株?方法:针对IL-15基因作用的功能域,设计了靶向IL-15基因Exon2的gRNA? 构建PX458-gRNA重组质粒并转化感受态细胞Stbl3,筛选出重组子后进行测序,通过测序确认了所设计的gRNA的有效性?并进一步通过流式细胞分选以及ELISA法检测筛选出的MGC-803胃癌细胞株IL-15基因的表达水平?结果:测序结果显示敲除质粒构建成功,与阴性对照组相比,转染PX458-IL-15-gRNA质粒组IL-15的表达水平明显低于阴性对照组,差异具有统计学意义(P < 0.001)?结论:利用CRISPR-Cas9系统成功构建了IL-15基因敲除的MGC-803 细胞,为后续研究IL-15在肿瘤中的作用机制和功能奠定了基础?

关 键 词:IL-15  CRISPR/Cas9系统  基因敲除
收稿时间:1/3/2016 12:00:00 AM

Constration of IL-15 knockout MGC-803 cell line by CRISPR/Cas9 technology
Yu Jing,Sun Li,Sun Xiaoxian,Cheng Zhihong,Qian Hui,Xu Wenrong and Zhu Wei.Constration of IL-15 knockout MGC-803 cell line by CRISPR/Cas9 technology[J].Acta Universitatis Medicinalis Nanjing,2016(2):140-143.
Authors:Yu Jing  Sun Li  Sun Xiaoxian  Cheng Zhihong  Qian Hui  Xu Wenrong and Zhu Wei
Institution:Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013,Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013,Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013,Department of General Surgery,the Affiliated People Hospital of Jiangsu university,Zhenjiang 212001,China,Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013,Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013; Department of Clinical Labortories,the Affiliated Hospital of Jiangsu university,Zhenjiang 212001 and Department of Clinical Labortories,School of Medical Technology,Jiangsu University,Zhenjiang 212013
Abstract:Objective:To construct IL-15 knockout MGC-803 cells by using CRISPR/Cas9 genome engineering technology. Methods:Guid RNA was designed by trageting the second exon of IL-15 gene,which encoded its homeodomains. PX458-gRNA recombinant plasmid was constructed and transformed into competent Stbl3. To confirm the efficitiveness of designed gRNA,the recombinan was screened and sequenced. The expression of IL-15 gene of MGC-803 cells sorted through FACS was detected by ELISA, and the cell cycle was detected by FACS. Results:Compared with negative control group,the expression of IL-15 transfected with PX458-IL-15-gRNA plasmid was significantly lower (P < 0.001). Conclusion:The IL-15 konctout gastric cancer cell line(MGC-803 cells)had been successfully constructed by using CRISPR/Cas9 system,which lays the foundation for further study of the mechanism and founction of IL-15 in tumors.
Keywords:IL-15  CRISPR/Cas9 system  gene knockdown
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