CRISPR/Cas9系统介导的EZH2基因定点敲入Hut78细胞系的构建  

Construction of EZH2 gene site-directed knock-in Hut78 cells by CRISPR/Cas9 system

作　　者：鲁卓林[1] 熊显佳 吴云丹[3] 周慧[4] 贾军[5] 王双林[6] 武莉莉[2] 刘艺洁[2] 乔阳[2] 杨冰[2] 赵秀娟[2] 王青松[2] 韩春勇[7] 张玲[2] 孙琰[2] LU Zhuo-lin;XIONG Xian-jia;WU Yun-dan;ZHOU Hui;JIA Jun;WANG Shuang-lin;WU Li-li;LIU Yi-jie;QIAO Yang;YANG Bing;ZHAO Xiu-juan;WANG Qing-song;HAN Chun-yong;ZHANG Ling;SUN Yan(Department of Pharmacy, Tianjin Children’s Hospital, Tianjin 300134, China;Basic Medical College of Tianjin Medical University;Department of Endocrinology, The 254th Hospital of the Chinese People’s Liberation Army;College of Pharmacy of Tianjin Medical University;Ankle Two Units, Tianjin Hospital;Department of Cardiac and Thoracic Surgery,Tianjin Medical University General Hospital;Department of Breast Reconstruction, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy)

机构地区：[1]天津市儿童医院药剂科,300134 [2]天津医科大学基础医学院 [3]中国人民解放军第二五四医院内分泌科 [4]天津医科大学药学院 [5]天津医院足踝二病区 [6]天津医科大学总医院心胸外科 [7]天津医科大学肿瘤医院乳房再造科,国家肿瘤临床医学研究中心,肿瘤防治重点实验室

出　　处：《天津医药》2017年第5期449-453,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81500170,81472052);天津市自然科学基金项目(15JCYBJC25400,16JCQNJC12100,16JCQNJC10300);天津市高等学校科技发展基金计划项目(20130103);教育部留学回国人员科研启动基金资助项目;天津医科大学肿瘤医院科研项目(B1516)

摘　　要：目的利用CRISPR/Cas9系统构建定点敲入EZH2基因的Hut78细胞系。方法构建EZH2表达载体pMD-18T-EZH2和单向导RNA(sg RNA)表达载体pSpCas9(BB)-2A-Puro-sg RNA,将两个载体共转染Hut78细胞,通过qPCR检测EZH2 mRNA的表达情况,通过Western Blot检测EZH2和H3K27me3蛋白的表达情况。结果测序结果显示,构建的pMD-18T-EZH2和pSpCas9(BB)-2A-Puro-sg RNA表达载体插入序列完全正确。将构建的两个质粒共转染Hut78细胞,qPCR结果显示EZH2基因在RNA水平上表达显著上调;Western blot结果显示EZH2和H3K27me3蛋白表达水平显著提高。结论通过CRISPR/Cas9系统成功构建了定点敲入EZH2基因的Hut78细胞系。Objective To construct the Hut78cell line with EZH2gene knocked into by CRISPR/Cas9system.Methods The EZH2expression vector pMD-18T-EZH2with homologous arm and the sgRNA expression vector pSpCas9(BB)-2A-Puro-sgRNA,which could cut the double stranded genomic DNA,were constructed,and the two vectors were cotransfectedinto Hut78cells.Then the expression of EZH2mRNA was detected by qPCR,and the expressions of EZH2andH3K27me3proteins were detected by Western blot assay.Results The pMD-18T-EZH2and pSpCas9(BB)-2A-PurosgRNArecombinant vectors were confirmed by DNA sequencing.When Hut78cells were transfected with the tworecombinant plasmid,qPCR results showed that the expression of EZH2mRNA was significantly increased,and Western blotanalysis showed that the expressions of EZH2and H3K27me3proteins were significantly increased.Conclusion EZH2gene is successfully knocked into Hut78cells by CRISPR/Cas9system.

关 键 词：甲基化 EZH2基因 CRISPR/Cas9n系统 基因编辑 

分 类 号：R349.69[医药卫生—基础医学] R349.83