摘要
在人源HIF-1α的PAS结构域内设计特异性靶点,构建Cas9-gRNA-HIF-1α敲除质粒,并将其转染至细胞后经嘌呤霉素筛选出21个单克隆细胞株,随后通过酶切、测序分析及下游基因验证等方法检测,最后得到一株特异性敲除HIF-1α的纯合子细胞(H20)。所设计的靶点能够有效地编辑HIF-1α,将为在其他细胞中通过CRISPR/Cas9系统特异性敲除HIF-1α提供方法借鉴。与此同时,也将为后续揭示HIF-1在由正常排卵事件演变成卵巢肿瘤过程中的作用机制奠定基础。
A specific target site obtained at the PAS (Per-ARNT-Sim) domain of human HIF-1α was used to construct a Cas9-gRNA- HIF-1α knockout plasmid. After transfected it into KGN cells, 21 monoclonal cell lines were selected by puromycin, and then determined by enzyme digestion, sequencing analysis and downstream gene verification, and finally obtained a specifically knockout HIF-1α homozygous cells (H20). Collectively, the specific knockout site used in this study can be efficiently target against HIF-1α , which will provide a reference for the specific knockout of HIF-1α in other cells through CRISPR/Cas9 system.
作者
杨芳
张宝云
向伟
王凭青
向远彩
YANG Fang;ZHANG Baoyun;XIANG Wei;WANG Pingqing;XIANG Yuancai(School of Basic Medical Sciences,Southwest Medical University,Luzhou 646000,China;Bioengineering College,Chongqing University,Chongqing 400000,China)
出处
《生物学杂志》
CAS
CSCD
北大核心
2020年第6期33-37,共5页
Journal of Biology
基金
西南医科大学校级项目(2018-ZRQN-108)
四川省科技厅项目(2019YJ0482)
泸州市-西南医科大学联合项目(2019LZXNYDZ03)。
