基于CRISPR/Cas9技术的人胚胎干细胞多模态示踪系统构建  

The Construction of Multimodality Imaging System in Human Embryonic Stem Cells Based on CRISPR/Cas9

作　　者：路兴爱 吴洁[1,2] 赵振奥[1,2] 胡士军[1,2] LU Xingai;WU Jie;ZHAO Zhen＇ao;HU Shijun(Institute for Cardiovascular Science,Soochow University,Suzhou Jiangsu 215000,China;Department of Cardiovascular Surgery,First Affiliated Hospital of Soochow University,Suzhou Jiangsu 215006,China)

机构地区：[1]苏州大学心血管病研究所,江苏苏州215000 [2]苏州大学附属第一医院心脏大血管外科,江苏苏州215006

出　　处：《复旦学报（自然科学版）》2018年第5期667-674,共8页Journal of Fudan University：Natural Science

基　　金：国家重点研发计划(2017YFA0103700);国家自然科学基金(91739106;81770257;81500277);江苏省自然科学基金(BK20170002;BK20150345;17KJA310006);国家临床重点专科心血管外科;江苏省医学重点学科(实验室);江苏省临床医学研究中心的平台资助

摘　　要：干细胞治疗的关键是移植细胞可以在病变部位定植存活,通过多模态成像对移植细胞进行体内实时、定量的示踪,对于优化移植策略至关重要.其中,生物发光、PET成像和荧光成像是非常有效的多模态成像手段.为了实现多模态成像,我们采用自裂解多肽2A连接的方式,将3种成像技术对应的3个报告基因(Fluc、HSVttk、copGFP)同步表达.利用CRISPR/Cas9技术,以腺相关病毒整合位点1(Adeno-Associated Virus integration Site 1,AAVS1)为靶点,通过同源重组,在人胚胎干细胞H7中建立了不同启动子驱动多模态成像系统.我们发现CAG(Cytomegalovirus-immediate early enhancer/chickenβ-Actin/rabbitβ-Globin)启动子驱动的报告系统明显优于UB(Ubiquitin)启动子系统,且无沉默现象.The survival and retention are important for stem cell therapy.Multimodality imaging is powerful for tracking and quantifing the transplanted stem cells,providing a reliable way to optimize transplantaion strategy,including bioluminescence,PET imaging and Green Fluorescent Protein.In this study,Fluc,HSCttk and copGFP were constructed using 2 Apeptide fusion method for coexpression.By CRISPR/Cas9 technology,the three reporters drived by different promoters were targeted to AAVS1 site through homologous recombination.We found that the CAG（cytomegalovirus-immediate early enhancer/chickenβ-actin/rabbitβ-globin）promoter is more efficent to drive three reportors expression than Ubiquitin promoter,without gene silencing.

关 键 词：人胚胎干细胞 多模态成像 启动子 报告基因 

分 类 号：Q812[生物学—生物工程]