强力霉素诱导型Cas9基因编辑小鼠胚胎干细胞系的构建  

Construction of Dox inducible Cas9-genetic editing mouse embryonic stem cell line

作　　者：姚玉琛 朱少华 曹佳妮 张琳 杨月伟[1] 赵同标 YAO Yu-chen;ZHU Shao-hua;CAO Jia-ni;ZHANG Lin;YANG Yue-wei;ZHAO Tong-biao(College of Life Sciences,Qufu Normal University,Shandong,Qufu 273165,China;State Key Laboratory of Stem Cell and Reproductive Biology,Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]曲阜师范大学生命科学学院,山东曲阜273165 [2]中国科学院动物研究所干细胞与生殖生物学国家重点实验室,北京100101

出　　处：《发育医学电子杂志》2018年第3期160-165,共6页Journal of Developmental Medicine (Electronic Version)

基　　金：国家自然科学基金国际(地区)合作研究与交流项目-重点国际(地区)合作研究项目(31720103907);科技部重点研发计划(2018YFA0108402);中国科学院A类战略性先导科技专项(XDA16030302)

摘　　要：目的建立强力霉素(doxmycin,Dox)诱导型Cas9基因编辑小鼠胚胎干细胞系,以满足不同条件下基因编辑的实验需求。方法根据四环素(tetracycline,Tet)-on诱导表达调控系统的原理,分别构建含有调控元件反义Tet转录激活因子(reverse tetracy-cline transcriptional activator,rt TA)的表达质粒p CDH-CAG-rt TA-IRES-m Cherry和含有Tet应答元件(Tet-responsive element,TRE)及Cas9的表达质粒p Tight-Cas9-IRES-GFP-Tight-Puro,其中红色荧光蛋白m Cherry通过内部核糖体进入位点(internal ribosome entry site,IRES)与rt TA表达,绿色荧光蛋白(green fluorescent protein,GFP)通过IRES与Cas9相连,分别指示rt TA与Cas9的表达,Puro抗性基因作为药物筛选标记。将上述两种质粒包装为慢病毒,分步转入小鼠胚胎干细胞中,建立Dox诱导Cas9表达的小鼠胚胎干细胞系。结果首先通过瞬时转染方法在293T细胞中验证其可诱导性,然后结合报告荧光与药物筛选获得同时整合有调控元件rt TA与应答元件TRE及Cas9的小鼠胚胎干细胞系,该细胞系加入Dox后出现有GFP报告荧光表达,实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,rt-q PCR)结果同时表明Cas9可以被成功诱导表达。结论本研究成功构建了Dox诱导Cas9表达的小鼠胚胎干细胞系,该细胞系在基因编辑方法上更加灵活可控,具有很高的潜在应用价值。Objective To establish Doxmycin（Dox） inducible Cas9-genetic editing mouse embryonic stem cell（m ESC） line to meet various research requirements in gene editing under different conditions. Methods According to the principle of the tetracycline（Tet）-on inducible expression regulatory system, the expression plasmid（p CDH-CAG-rt TA-IRES-m Cherry） of containing regulatory element reverse tetracycline transcriptional activator（rt TA）, and the expression plasmid（p Tight-Cas9-IRES-GFP-Tight-Puro） of containing Tet-responsive element（TRE） and Cas9 coding sequence, were constructed respectively. The red fluorescent protein m Cherry was ligated with internal ribosome entry site IRES in the rt TA, and green fluorescent protein（GFP） was ligated with IRES in the Cas9, indicating the expression of rt TA and Cas9. Puro resistant gene was constructed for drug selection. The above plasmids were packaged into lentivirus and transfected into m ESC in stages, then constructed Dox inducible Cas9-genetic editing m ESC Line. Results Firstly, the inducibility of this system was verified by 293 T transient transfection assay. Then, the m ESC line with both rt TA and TRE-Cas9 integration were established by combination with report fluorescence and drug selection which appeared the expressed of GFP report after Dox addition. Quantitative polymerase chain reaction assay also showed that Cas9 expression could be successfully induced by Dox. Conclusion In this study, a Dox inducible Cas9-genetic editing m ESC Line has been successfully constructed, which is more flexibility and controllability in gene editing, and holds high potential application in the future.

关 键 词：CRISPR/Cas9 四环素诱导 条件性敲除 小鼠ES细胞系 

分 类 号：Q78[生物学—分子生物学]