纯合敲除Cyp26s基因胚胎干细胞模型的建立  

The Establishment of Cyp26s Gene Homozygous Knockout Model for Embryonic Stem Cell

作　　者：曹欢欢 戴思雨 包美玲 李婧雅 张遵义 黄华荣 CAO Huanhuan;DAI Siyu;BAO Meiling;LI Jingya;ZHANG Zunyi;HUANG Huarong(School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China;College of Medicine and Life Science, Jiujiang University, Jiujiang 332000, China)

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036 [2]九江学院药学与生命科学学院,江西九江332000

出　　处：《杭州师范大学学报（自然科学版）》2018年第1期65-71,共7页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：国家自然科学基金项目(31101046);浙江省自然科学基金项目(LY15C070005);杭州农业科研主动设计项目(20162012A07)

摘　　要：基因敲除的胚胎干细胞模型在基因功能的研究中起着重要作用.为了建立一套在胚胎干细胞中简便高效纯合敲除基因的方法,以Cyp26s基因家族为例,应用CRISPR-Cas9基因修饰技术,对靶向gRNA设计、细胞筛选、阳性克隆鉴定等过程进行了优化.结果表明,通过双位点靶向gRNA设计,转染线性化的质粒,G418筛选7d,sgRNA位点外侧设计鉴定引物等策略,在4周内获得了3株纯合敲除Cyp26s基因(Cyp26a1^(-/-)、Cyp26b1^(-/-)和Cyp26c1^(-/-))的ES细胞系.Gene knockout embryonic stem cells model plays an important role in the study of gene function.In order to establishment a simple and efficient method to homozygous knockout gene in embryonic stem cells,the paper took Cyp26s gene families as an example;utilized CRISPR-Cas9genetic modification technology and optimized the related process,such as the design of targeted gRNA,the strategy of cell selection,positive cloning identification.The results showed that three ES cell lines of homozygous knockout Cyp26s gene(Cyp26a1-/-,Cyp26b1-/-or Cyp26c1-/-,respectively)were efficiently generated in4weeks by double gRNA sites design,linearized plasmid transfection,G418screening for7days,identification primer outside sgRNA site design strategy.

关 键 词：CRISPR-Cas9 Cyp26s 基因敲除 胚胎干细胞 

分 类 号：Q291[生物学—细胞生物学]