同济大学学报（医学版）2018，Vol.39Issue(2)：26-31,6.DOI:10.16118/j.1008-0392.2018.02.006
CRISPR/Cas9构建TGFBI稳定敲除HSC-3细胞系
Construction of TGFBI stable knockout HSC-3 cell line by CRISPR/Cas9 gene editing technique
摘要
Abstract
Objective To establish a TGFBI stable knockout oral squamous cell carcinoma(OSCC) cell line HSC-3 by CRISPR/Cas9 gene editing technique. Methods The 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. Results The 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). Conclusion The 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.关键词
CRISPR/Cas9/TGFBI/HSC-3细胞/基因敲除Key words
CRISPR/Cas9/TGFBI/HSC-3cell/gene knockout分类
医药卫生引用本文复制引用
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):26-31,6.基金项目
国家自然科学基金(81000438) （81000438）
上海市自然科学基金(14ZR1443600) （14ZR1443600）
