军事医学2017，Vol.41Issue(2)：96-100,105,6.DOI:10.7644/j.issn.1674-9960.2017.02.004
基于CRISPR/Cas9基因编辑技术的RIP1稳定敲除细胞模型的构建及功能研究
Construction and characterization of RIP1 stable knockout HaCaT cell line based on CRISPR/Cas9 system
摘要
Abstract
Objective To establish a stable RIP1 gene knockout cell line using CRISPR/Cas9 system in immortalized human epidermal cell line HaCaT,and to explore the function of RIP1 in HaCaT cells by this novel HaCaTRIP1KO cell line. Methods The small guide RNA(sgRNA)sequences targeting different exons of RIP1 designed according to sequence in GenBank were cloned into the SpCas9-2A-Puro V2.0 vectors to construct the recombinant PX459-sgRNA plasmid for RIP1 gene knockout.The HaCaT cells transfected with this recombinant plasmid were selected by puromycin to obtain the positive clone cells.Then,the monoclones with optimal gene knockout effect were further screened out by Western blotting and sequencing.CCK8 assay was performed to investigate the effect of RIP1 knockout on proliferation of HaCaT cells.The TNF-α-induced cell death of HaCaTWT cells and HaCaTRIP1KO cells was detected by flow cytometry,and the type of cell death was further determined using the caspase inhibitor Z-VAD-FMK.Results and Conclusion The HaCaTRIP1KO cell line was successfully constructed using CRISPR/Cas9 system.RIP1 knockout significantly reduced the proliferation of HaCaT cells. Besides,the HaCaTRIP1KO cells were more vulnerable than HaCaTWT cells to the TNF-α-induced cell death,which was dramatically rescued by Z-VAD-FMK,indicating that the process is caspase-dependent apoptosis in HaCaTRIP1KO cells.This work may contribute to the study of the functions of RIP1 in skin lesions.关键词
CRISPR/Cas9系统/基因敲除/受体相互作用丝/苏氨酸蛋白激酶1/HaCaT细胞/凋亡/细胞增殖Key words
CRISPR/Cas9 system/gene konckout/RIP1/HaCaT cell/apoptosis/cell proliferation分类
生物科学引用本文复制引用
陈瑶,刘青,邢微微,陈惠华,张冬冬,王园园,邹民吉,徐东刚,刘中成..基于CRISPR/Cas9基因编辑技术的RIP1稳定敲除细胞模型的构建及功能研究[J].军事医学,2017,41(2):96-100,105,6.基金项目
国家科技重大专项"重大新药创制"资助项目(2012ZX09102301-017) （2012ZX09102301-017）
河北省研究生创新资助项目(S2016020) （S2016020）
河北省自然科学基金资助项目(H2013201128,H2016201121) （H2013201128,H2016201121）
