军事医学2016，Vol.40Issue(4)：299-303,5.DOI:10.7644/j.issn.1674-9960.2016.04.008
Apak稳定敲除细胞系的建立及其p53活性和凋亡水平的变化
Generation of Apak stably knockout cell lines and variation of their p53 activity and apoptosis
摘要
Abstract
Objective To establish an Apak gene stable and permanent knockout cell line using CRISPR/Cas9 system in human colon cancer cells ( HCT116 cells), and study the effect of Apak knock-out on p53 activity and apoptosis. Methods The lentiCRISPR v2-sgRNA Apak expression plasmid was co-transfected with lentivirus coated plasmids pSPAX2 and pMD2.G.The supernatant was collected, filtered, and used to infect HCT116 cells.The positive clones were screened out by puromycin culture and Western blot was used to detect Apak knockout cell lines.Luciferase reporter gene assay, flow cytometry analysis and colony formation assay were used to examine p53 activity and apoptosis of Apak knockout cells, respectively.Results Apak knockout HCT116 cell lines were generated in which p53 activity and apoptosis were increased,but the colony formation was decreased.Conclusion The Apak stable knockout cell lines of HCT116 are successfully generated by CRISPR/Cas9 system for further functional study.关键词
慢病毒属/Apak/CRISPR/Cas9/敲除/基因,p53/质粒/凋亡/克隆形成Key words
Lentivirus/Apak/CRISPR/Cas9/knockout/genes,p53/plasmids/apoptosis/colony formation分类
生物科学引用本文复制引用
梅兴沙,王建,郑惠珍,田春艳..Apak稳定敲除细胞系的建立及其p53活性和凋亡水平的变化[J].军事医学,2016,40(4):299-303,5.基金项目
国家自然科学基金资助项目（31540072，31270799）；国家科技合作专项资助项目（2014DFB30020）；北京市自然科学基金资助项目（5142019）；蛋白质组学国家重点实验室开放课题 （）
