Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology.
10.3779/j.issn.1009-3419.2018.05.02
- VernacularTitle:利用CRISPR/Cas9慢病毒系统构建肺部EZH2基因敲除小鼠
- Author:
Fanrong MENG
1
;
Dan ZHAO
2
;
Qinghua ZHOU
1
;
Zhe LIU
2
Author Information
1. Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
2. Tianjin Medical University, Tianjin 300070, China.
- Publication Type:Journal Article
- Keywords:
CRISPR-Cas9;
EZH2;
Gene knockout;
sgRNA
- MeSH:
Animals;
CRISPR-Cas Systems;
Enhancer of Zeste Homolog 2 Protein;
genetics;
metabolism;
Female;
Gene Knockout Techniques;
Gene Targeting;
Humans;
Lung Neoplasms;
genetics;
metabolism;
Male;
Mice;
Mice, Inbred C57BL;
Mice, Knockout;
RNA, Guide
- From:
Chinese Journal of Lung Cancer
2018;21(5):358-364
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: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 using 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 efficiency 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 results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently 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 decreased in the mouse lung tissue.
CONCLUSIONS: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.
