利用CRISPR/Cas9慢病毒系统敲除人源HOIP基因

泛素化修饰是一种能调节诸多细胞功能的重要蛋白质翻译后修饰,近几年研究发现了一种新的泛素化修饰即线性泛素化修饰,LUBAC(linear ubiquitin chain assembly complex)是目前已知唯一的导致蛋白质发生线性泛素化修饰的E3酶。目前LUBAC复合物的底物和功能所知甚少。为了探索LUBAC复合物新的底物和功能,利用CRISPR/Cas9慢病毒系统构建稳定敲除LUBAC核心组分HOIP基因的HeLa细胞系,通过质谱鉴定此细胞系和对照细胞的差异泛素化修饰蛋白质,即可能是LUBAC新的底物。首先将靶向HOIP基因的不同CRISPR序列插入lenti CRISPRv2载体中,制备慢病毒质粒感染He La细胞,然后使用嘌呤霉素压力筛选,用Western Blot方法检测HOIP基因的敲除效果,最后成功地构建HOIP基因稳定敲除HeLa细胞系。此稳定细胞系的构建为日后深入研究LUBAC的功能提供一个人类细胞模型。

Knocking Out Human HOIP Gene by CRISPR/Cas9 Lentiviral System

Ubiquitination is an important post-translational modification involved in the regulation of a broad variety of cellular functions. In recent years, a new ubiquitin linkage type named linear ubiquitination has been identified. So far LUBAC(Linear ubiquitin chain assembly complex) is the only identified E3 to generate linear chains, however, little is known about its substrates and functions. To investigate the new substrates and functions of LUBAC, we established a stable HeLa cell line with knocking out HOIP gene which is the core component of LUBAC through CRISPR/Cas9 lentivirus system.We firstly integrated different CRISPR sequences targeting HOIP geneinto lentiCRISPR v2vector, making HeLa cellstransfected with the lentiviral expression plasmid and then got the anti-puro positive cells under puromycin. The knockout efficiency of HOIP gene was detected via Western Blot.Finally the stable HeLa cell line was established,which would provide a human cell model for further study of functions of LUBAC in the future.