Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology

Chinese Journal of Lung Cancer

Qinghua ZHOU¹  Fanrong MENG¹  Dan ZHAO²  Zhe LIU² 

[1] Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China;Tianjin Medical University, Tianjin 300070, China;

关键词: CRISPR-Cas9;    EZH2;    sgRNA;    Gene knockout;   

DOI  :  10.3779/j.issn.1009-3419.2018.05.02

来源: DOAJ

【 摘 要 】

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 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. 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.

【 授权许可】

Unknown