应用CRISPR/Cas9技术构建IL-15敲除的MGC-803胃癌细胞株  

Constration of IL-15 knockout MGC-803 cell line by CRISPR / Cas9 technology

作　　者：俞静[1] 孙丽[1] 孙晓仙 陈志红[2] 钱晖[1] 许文荣[1,3] 朱伟[1] 

机构地区：[1]江苏大学医学院检验系,江苏镇江212013 [2]江苏大学附属人民医院外科,江苏镇江212001 [3]江苏大学附属医院检验科,江苏镇江212001

出　　处：《南京医科大学学报（自然科学版）》2016年第2期140-143,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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-g RNA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。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-g RNA 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.

关 键 词：IL-15 CRISPR/Cas9系统 基因敲除 

分 类 号：R735.2[医药卫生—肿瘤]