| CRISPR/Cas9构建TGFBI稳定敲除HSC-3细胞系 |
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| 引用本文： | 汪丙杰,郑赛巍,马聪聪,史聪,李玉婷,何园.CRISPR/Cas9构建TGFBI稳定敲除HSC-3细胞系[J].同济大学学报(医学版),2018,39(2):26-31. |
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| 作者姓名： | 汪丙杰 郑赛巍 马聪聪 史聪 李玉婷 何园 |
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| 作者单位： | 同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072,同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072,同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072,同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072,同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072,同济大学口腔医学院，上海 200072；同济大学附属口腔医院黏膜病学教研室-上海牙修复与再生工程实验中心，上海200072 |
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| 基金项目： | 国家自然科学基金（81000438）；上海市自然科学基金（14ZR1443600） |
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| 摘 要： | 目的利用CRISPR/Cas9技术稳定敲除人口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）细胞系HSC-3中的TGFBI基因。方法根据CRISPR/Cas9靶点设计原则，设计能特异性针对于TGFBI外显子3上下游的小向导RNA（small guide RNA, sgRNA），并以PX330质粒为骨架构建能表达此sgRNA的真核重组质粒。将此质粒以及PGK-puro质粒共转染HSC-3细胞，用嘌呤霉素抗性筛选细胞克隆，并用PCR、核酸电泳和测序初步确定基因敲除情况，再通过实时荧光定量PCR及免疫印迹法确认细胞中TGFBI的mRNA和蛋白水平的表达变化情况。结果通过CRISPR/Cas9技术，HSC-3中的TGFBI基因外显子3的核苷酸序列已被完全敲除。免疫印迹法检测显示，相对于HSC-3，TGFBI敲除细胞中无法检测到TGFBI蛋白的表达。同时，通过RT-qPCR对部分基因进行检测，发现一系列与细胞周期和上皮间充质转化相关基因表达发生了改变（P＜0.05）。结论通过CRISPR/Cas9技术成功获得了TGFBI基因敲除的HSC-3细胞系，为进一步研究TGFBI在OSCC中的作用及其机制提供了理论基础。
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| 关 键 词： | CRISPR/Cas9 TGFBI HSC-3细胞 基因敲除 |
| 收稿时间： | 2017/11/21 0:00:00 |
Construction of TGFBI stable knockout HSC-3 cell line by CRISPR/Cas9 gene editing technique |
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| WANG Bing-jie,ZHENG Sai-wei,MA Cong-cong,SHI Cong,LI Yu-ting and HE Yuan.Construction of TGFBI stable knockout HSC-3 cell line by CRISPR/Cas9 gene editing technique[J].Journal of Tongji University(Medical Science),2018,39(2):26-31. |
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| Authors: | WANG Bing-jie ZHENG Sai-wei MA Cong-cong SHI Cong LI Yu-ting and HE Yuan |
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| Institution: | School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China,School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China,School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China,School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China,School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China and School of Stomatology, Tongji University, Shanghai 200072, China; Dept. of Oral Medicine, School and Hospital of Stomatology, Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China |
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| Abstract: | |
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| Keywords: | CRISPR/Cas9 TGFBI HSC-3cell gene knockout |
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