| 引用本文： |
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汪丙杰,郑赛巍,马聪聪,等.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. [点击复制]
- WANG Bing-jie,ZHENG Sai-wei,MA Cong-cong,et al.Construction of TGFBI stable knockout HSC-3 cell line by CRISPR/Cas9 gene editing technique[J].同济大学学报（医学版）,2018,39(2):26-31. [点击复制]
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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。 |
| 关键词: CRISPR/Cas9 TGFBI HSC-3细胞 基因敲除 |
| DOI：10.16118/j.1008-0392.2018.02.006 |
| 投稿时间：2017-11-21 |
| 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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| Construction of TGFBI stable knockout HSC-3 cell line by CRISPR/Cas9 gene editing technique |
| WANG Bing-jie,ZHENG Sai-wei,MA Cong-cong,SHI Cong,LI Yu-ting,HE Yuan |
| (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) |
| Abstract: |
| ObjectiveTo establish a TGFBI stable knockout oral squamous cell carcinoma (OSCC) cell line HSC-3 by CRISPR/Cas9 gene editing technique. MethodsThe small guide RNA （sgRNA）sequences targeting specifically to the upstream and the downstream of TGFBI exon 3 were designed according to the principle of CRISPR/Cas9 target design and cloned into PX330 plasmid to construct a eukaryotic recombination plasmid expressing the sgRNA. The plasmids and PGK-puro plasmid were co-transfected into HSC-3 cells and the cells were selected by puromycin. The gene knockout was detected by PCR, nucleic acid electrophoresis and sequencing, mRNA and protein expression of TGFBI in the cells was detected by RT-qPCR and Western blotting, respectively. ResultsThe exon3 nucleotide sequence of TGFBI was deleted in HSC-3 by CRISPR/Cas9 gene editing technique. The result of Western blotting indicated that TGFBI protein expression was not detected in TGFBI knockout cells. In addition, RT-qPCR showed that the expression of a series of genes involving in cell cycle and epithelial-mesenchymal transition were changed after TGFBI knockout（P＜0.05）. ConclusionThe TGFBI stable knockout HSC-3 cell line has been constructed successfully by CRISPR/Cas9 gene editing technique, which would be used for further study on the function and mechanism of TGFBI in OSCC. |
| Key words: CRISPR/Cas9 TGFBI HSC-3cell gene knockout |
