中国肺癌杂志2018，Vol.21Issue(5)：358-364,7.DOI:10.3779/j.issn.1009-3419.2018.05.02
利用CRISPR/Cas9慢病毒系统构建肺部EZH2基因敲除小鼠
Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology
孟凡荣 1赵丹 2周清华 1刘喆2
作者信息
- 1. 300052 天津,天津医科大学总医院,天津市肺癌研究所,天津市肺癌转移与肿瘤微环境实验室
- 2. 300070 天津,天津医科大学
- 折叠
摘要
Abstract
Background and objective It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA)at desired genomic loci.The aim of this study is that the animal model of EZH2 gene knockout was constructed us-ing CRISPR/Cas9 technology. Methods In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene.Then,their gene-targeting effciency were detected by SURVEYOR assay.The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR. Results The experimental re-sults of NIH-3T3 cells verify that the designed sgEZH2 can effciently effect the cleavage of target DNA by Cas9 in vitro.The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly de-creased in the mouse lung tissue. Conclusion The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.关键词
CRISPR/Cas9系统/EZH2/sgRNA/基因敲除Key words
CRISPR-Cas9/EZH2/sgRNA/Gene knockout引用本文复制引用
孟凡荣,赵丹,周清华,刘喆..利用CRISPR/Cas9慢病毒系统构建肺部EZH2基因敲除小鼠[J].中国肺癌杂志,2018,21(5):358-364,7.
