军事医学2017，Vol.41Issue(4)：265-268,4.DOI:10.7644/j.issn.1674-9960.2017.04.004
利用CRISPR/Cas9技术构建Nedd4基因敲除BMDM巨噬细胞系
Construction of Nedd4 knockout BMDM cell line by CRISPR/Cas9 technology
摘要
Abstract
Objective To construct Nedd4 knockout bone marrow-derived macrophages(BMDM) cell line by CRISPR/Cas9 technology and to provide an effective tool for studying the function and mechanism of Nedd4 in macrophage.Methods First,three high-grade sgRNAs targeting Nedd4 gene exons were screened using the online tool before synthesized sgRNAs were inserted into the PX330 plasmid respectively.Secondly,the recombinant plasmids were transferred into BMDM cells and monoclonal cells were obtained by limiting dilution method.The protein levels of NEDD4 in monoclonal cells were detected by Western blotting.Finally,the DNA sequence of the monoclonal cells was confirmed by sequence analysis.Results One Nedd4 knockout BMDM cell line was obtained.The sequencing result showed that the Nedd4 gene had 16bp deletion mutation in this cell line.Conclusion The Nedd4 knockout BMDM macrophage cell line constructed by CRISPR/Cas9 technology will be a useful tool for studying the function and mechanism of Nedd4 in BMDM cells.关键词
CRISPR/Cas9技术/Nedd4/基因敲除/巨噬细胞Key words
CRISPR/Cas9 gene editing technology/Nedd4/gene knockout/macrophages分类
生物科学引用本文复制引用
张市会,周虹,刘庆军..利用CRISPR/Cas9技术构建Nedd4基因敲除BMDM巨噬细胞系[J].军事医学,2017,41(4):265-268,4.基金项目
国家自然科学基金青年基金资助项目(81600148) （81600148）
