运用CRISPR/Cas9技术构建敲除体轴抑制蛋白1(AXIN1)基因的ACT-1人未分化甲状腺癌细胞系  

Construction of ACT-1 human undifferentiated thyroid cancer cell line with knockout of axis inhibition protein 1 (AXIN1) gene using CRISPR/Cas9

作　　者：文丹 黄蓉 谢建平[1] 温琥玲[1] 林师宇[1] WEN Dan;HUANG Rong;XIE Jianping;WEN Huling;LIN Shiyu(Department of Nuclear Medicine,Affiliated Hospital of North Sichuan Medical College,Nanchong 637000,China;Department of Pharmacy,North Sichuan Medical College,Nanchong 637000,China)

机构地区：[1]川北医学院附属医院核医学科,四川南充637000 [2]川北医学院药学院,四川南充637000

出　　处：《细胞与分子免疫学杂志》2020年第5期419-424,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：市校科技战略合作专项(NSMC 20170435);川北医学院附属医院院级科研课题(2020JC004)。

摘　　要：目的构建体轴抑制蛋白1(AXIN1)基因敲除的ACT-1人未分化甲状腺癌单克隆细胞系。方法用分子克隆技术及成簇的规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)基因编辑技术构建AXIN1基因敲除的单克隆细胞系;采用实时荧光定量PCR检测ACT-1细胞的AXIN1 mRNA水平,Western blot法检测AXIN1蛋白水平。结果通过T7检测成功获得有效的两个单链导向RNA(sgRNA)Cr3及Cr5;通过酶切鉴定及测序成功构建携带绿色荧光蛋白(GFP)的靶向AXIN1的sgRNA病毒载体;成功获得包括阴性对照在内的4个单克隆ACT-1未分化甲状腺癌细胞系;敲除组细胞AXIN1 mRNA及蛋白水平均明显降低。结论采用CRISPR-Cas9技术成功建立了敲除AXIN1基因的ACT-1未分化甲状腺癌细胞系。Objective To construct the axis inhibition protein 1(AXIN1)gene-knockout ACT-1 human undifferentiated thyroid cancer single clone cell line.Methods Molecular cloning technology and clustered regularly interspaced short palindromic repeats/Cas9 nuclease(CRISPR/Cas9)were used to construct AXIN1 gene-knockout single clone cell lines.Real-time quantitative PCR and Western blotting were used to detect AXIN1 mRNA and protein levels of ACT-1 cells,respectively.Results T7 detection results showed two effective single guide RNAs(sgRNAs)Cr3 and Cr5 were successfully constructed;enzyme digestion identification and sequencing showed AXIN1-targeted sgRNA viral vectors carrying green fluorescent protein(GFP)were successfully constructed.We successfully obtained 4 monoclonal ACT-1 undifferentiated thyroid cancer cell lines.AXIN1 mRNA and protein levels in the gene-knockout group were significantly reduced.ConclusionThe ACT-1 undifferentiated thyroid cancer cell line with AXIN1 gene knockout has been successfully constructed using CRISPR/Cas9.

关 键 词：成簇的规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9) 未分化甲状腺癌 体轴抑制蛋白1(AXIN1) 

分 类 号：R392.11[医药卫生—免疫学] R736.1[医药卫生—基础医学]