利用成簇规律间隔短回文重复序列及其相关蛋白9基因编辑技术快速构建GATA1-mCherry实时追踪人多能干细胞株的研究  

Research on quickly construct GATA1-mCherry to track human pluripotent stem cells lines in real time by CRISPR/Cas9 gene editing technology

作　　者：谢芳莘 蔡信平 周建华 李霞 赖默温 张勇刚 马峰 Xie Fangxin;Cai Xinping;Zhou Jianhua;Li Xia;Lai Mowen;Zhang Yonggang;Ma Feng(Institute of Blood Transfusion,Chinese Academy of Medical Sciences,Chengdu 610052,Sichuan Province,China)

机构地区：[1]中国医学科学院输血研究所,成都610052

出　　处：《国际输血及血液学杂志》2021年第6期520-529,共10页International Journal of Blood Transfusion and Hematology

摘　　要：目的探讨利用成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)9基因编辑技术快速构建GATA1-mCherry实时追踪人多能干细胞(hPSC)株的方法。方法①选择GATA1基因为目的基因,利用CRISPR/Cas9技术构建共表达载体[Cas9和向导RNA(gRNA)]和打靶载体(重组臂),并且以电穿孔的方式将共表达载体、打靶载体及环化重组酶(Cre)导入hPSC的H1细胞系。经电穿孔后的H1细胞培养2 d后,选择24个H1细胞单克隆进行PCR扩增,以验证共表达载体和打靶载体是否被成功导入,以及Cre是否成功去除H1细胞单克隆中的筛选标志物。②挑取经PCR验证的条带大小正确的阳性H1细胞单克隆,进行细胞培养后,提取H1细胞单克隆基因组,并且分别采用引物对1/2(GATA15′端臂外引物/mCherry 3′端引物)和3/5(HygroR 5′端引物/GATA13′端臂外引物)对基因组进行PCR扩增,以验证该H1细胞单克隆是否具有GATA1和mCherry正确序列。③使用构建完成的H1∶GATA1-mCherry细胞进行造血功能验证实验,以确认基因编辑是否改变H1细胞特性,以及造血分化能力。选择未经基因编辑H1细胞和H1∶GATA1-mCherry细胞,置于相同的造血分化培养条件培养14 d后,分别于610 nm激发波长免疫荧光显微镜下观察,并采用流式细胞术(FCM)对细胞表面标志物CD34、CD43、CD45进行检测。选取培养至GATA1大量表达时期的H1∶GATA1-mCherry细胞,进行细胞甩片和免疫荧光染色实验。对经过基因编辑的H1∶GATA1-mCherry细胞,进行GATA1特异抗体染色体,观察其能否准确追踪并指示GATA1。结果①经电穿孔转入打靶载体和共表达载体后,H1细胞单克隆PCR扩增产物的电泳结果显示,H1细胞经电穿孔后,可获得2710和945 bp的PCR扩增产物电泳条带;电泳条带为2710 bp,说明该克隆基因组成功敲入GATA1和mCherry序列,但是Cre没有去除筛选标志物;条带大小为945 bp,说明该克隆基因组成功敲入GATA1和mCherry序列,并且Cre�Objective To explore an effective method for quickly constructing GATA1-mCherry to track human pluripotent stem cells(hPSC)lines in real time by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)9 gene editing technology.Methods①GATA1 gene was taken as the target gene.Use CRISPR/Cas9 technique to construct co-expression vectors[Cas9 and guidance RNA(gRNA)]and target vectors(recombinant arm).The co-expression vectors,target vectors and cyclization recombination(Cre)were introduced into H1 cells by electroporation.After these electroporated H1 cells were cultured for 2 d,24 monoclonal H1 cells were selected for PCR test to verify whether co-expression vectors and target vectors were successfully introduced,and screening markers were successfully removed by Cre in monoclonal cells.②Positive monoclonal H1 cells with correct band size verified by PCR were selected and cultured for cells amplification.The genome of monoclonal H1 cells was extracted and PCR was performed respectively with primer pair 1/2(GATA15′-end primer/mCherry 3′-end primer)and 3/5(HygroR 5′-end primer/GATA13′-end primer)to verify whether the monoclonal H1 cells had correct sequences of GATA1 and mCherry.③The constructed H1∶GATA1-Cherry cells were used for hematopoietic function verification experiment to determine whether gene editing could alter intrinsic characteristics and hematopoietic differentiation of H1 cells.Both unedited H1 cells and H1∶GATA1-mCherry cells were cultured in the same hematopoietic differentiation conditions for 14 d,then cells were observed under 610 nm excitation wavelength immunofluorescence microscope and detected by flow cytometry(FCM)with CD34,CD43 and CD45 markers,respectively.③The H1∶GATA1-mCherry cells cultured to the period of abundant expression of GATA1 were used for cell rejection and immunofluorescence staining.The specific antibody of GATA1 was used to observe whether the H1∶GATA1-mCherry cells after gene editing could accurately track and

关 键 词：GATA1转录因子 多能干细胞 基因敲入技术 成簇规律间隔短回文重复序列 CRISPR相关蛋白 GATA1基因 

分 类 号：Q78[生物学—分子生物学]