利用CRISPR/Cas9方法敲除CBFβ基因及CBFβ真核表达载体的构建  

Construction of CBFβ Knockout Cell Line by CRISPR/Cas9 and the Construction of CBFβ Eukaryotic Expression Vector

作　　者：徐凤超 宋红肖[1] 谭广云[1] 陈建柱 

机构地区：[1]吉林大学第一医院转化医学研究院免疫学研究所,长春130061

出　　处：《科学技术与工程》2016年第18期135-139,共5页Science Technology and Engineering

基　　金：吉林省科技厅青年基金项目(20160520161JH)资助

摘　　要：构建CBFβ敲除Hep G2细胞系及CBFβ真核表达载体。CRISPR/Cas9基因敲除,将表达CBFβsgRNA,Cas9的表达载体共转Hep G2细胞,嘌呤霉素筛选基因敲除细胞,构建细胞系。infusion cloning方法构建CBFβ表达载体,Hep G2细胞提取RNA,反转成c DNA,设计CBFβ引物扩增目的基因,连接,转化,挑取阳性克隆,PCR鉴定,酶切鉴定,测序进一步鉴定。获得了稳定敲除CBFβ的Hep G2细胞系及在Hep G2细胞中稳定表达的CBFβ真核表达载体。为研究CBFβ基因在对乙型肝炎病毒复制的调控及调控机制奠定了基础。The CBFβknockout Hep G2 Cell Line and the HA-CBFβ eukaryotic expression vector was constructed. CRISPR/Cas9 gene knockout,Cas9 expression vector and CBFβ sgRNA were co-transfected into Hep G2 cell,puromysin was used for the positive gene knockout cell selection; Construct the CBFβ expression vector by infusion coloning method. RNA was extracted from the Hep G2 cells,and reversed to c DNA,specific PCR primers were designed to amplify the CBFβgene which was ligated to VR1012 vector,and transformated to Trans5 a competent cell,pick colonies for PCR and digestion verification,and finally conformed by sequencing. The CBFβ knockout Hep G2 cell line and the CBFβ eukaryotic expression vector which can stably expressed in Hep G2 cells have been established. A foundation for the study of CBFβ gene in regulating HBV replication was laied.

关 键 词：CBFβ CRISPR/Cas9 基因敲除 真核表达载体 

分 类 号：Q51[生物学—生物化学]