CRISPR/Cas9建立IL-12p35基因敲除大鼠C6细胞系表达研究  

Expression of IL-12p35 knock out rat C6 cell line by CRISPR/Cas9 technology

作　　者：孙瑾[1] 张俊卿[1] 黄延林[1] 杨芳裕[1] 董桂江 田新华[1] SUN Jin;ZHANG Jun-Qing;HUANG Yan-Lin;YANG Fang-Yu;DONG Gui-Jiang;TIAN Xin-Hua(Neurosurgery of Zhongshan Hospital Affiliated to Xiamen University,Xiamen 361004,China)

机构地区：[1]厦门大学附属中山医院神经外科

出　　处：《中国免疫学杂志》2019年第20期2452-2456,共5页Chinese Journal of Immunology

基　　金：福建省青年基金项目(2015-2-50);福建省科技卫生联合基金(2017J01367)

摘　　要：目的:运用CRISPR/Cas9技术敲除大鼠C6细胞系的IL-12p35基因,构建IL-12p35基因稳定敲除细胞株。方法:构建Lenti-sgRNA-EGFP质粒CRISPR/Cas9系统的Cas9核酸内切酶基因,慢病毒感染C6细胞后进行MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]检测及PI-FACS(Facial Action Coding System)细胞周期检测。结果:针对IL-12p35基因的KO1、KO2、KO3三个靶点,进行慢病毒转染,转染效率均大于80%。酶切前后靶点活性均下调,免疫印迹验证C6细胞中靶点有效性。MTT检测结果IL-12p35基因敲除后于第5天细胞增殖倍数明显降低。IL-12p35基因敲除后显著影响细胞周期G1期,G2/M期。结论:本研究构建IL-12p35基因的CRISPR/Cas9敲除系统,获得稳定的IL-12p35基因敲除细胞株,为后期IL-12p35与胶质瘤细胞的发生、发展机制方面研究提供基础。Objective:Stable cell lines were screened by CRISPR/Cas9(Clustered Regularly Interspersed Short Palindromic Repeats/Cas9) method detected by IL-12 p35 gene.Methods: The Cas9 endonuclease gene of the Lenti-sgRNA-EGFP plasmid CRIS PR/Cas9 System was constructed.MTT[3-(4-5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide and PI-FACS(Facial Action Coding System] cell cycle were detected after lentivirus infection with C6 cells.Results: IL-12 p35 gene three target lentivirus infection cells,fluorescence rate were more than 80%.Target activity was decreased before and after enzyme digestion,and immunoblot was used to verify the effectiveness of target in C6 cells.The results of MTT assay showed that IL-12 p35 gene knockout significantly decreased the cell proliferation multiple on day 5.After IL-12 p35 gene knockout,cell cycle G1 and G2/M were significantly affected.Conclusion: In this study,CRISPR/Cas9 knockout system of IL-12 p35 gene was constructed to obtain stable IL-12 p35 gene knockout cell lines,providing a basis for later studies on the occurrence and development mechanism of IL-12 p35 and glioma cells.

关 键 词：CRISPR/Cas9 C6 IL-12p35 慢病毒源性载体 

分 类 号：R730.3[医药卫生—肿瘤]