Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system
10.3760/cma.j.issn.1002-0098.2015.08.011
- VernacularTitle:CRISPR/Cas9系统在腺样囊性癌细胞中编辑纤连蛋白基因EDA片段的研究
- Author:
Yue YANG
1
;
Haicheng WANG
;
Shuyu XU
;
Jing PENG
;
Jiuhui JIANG
;
Cuiying LI
Author Information
1. 100081,北京大学口腔医学院·口腔医院中心实验室
- Keywords:
Carcinoma,adenoid cystic;
Knockout;
Clustered regularly interspaced short palindromic repeats/associated;
Extra domain A
- From:
Chinese Journal of Stomatology
2015;50(8):490-495
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells,via the clustered regularly interspaced short palindromic repeats (CRISPR)/associated proteins (Cas) system.Methods One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream.Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells.PCR and DNA sequence were used to testify the knockout cells,and the monoclones of EDA absent SACC cells were selected (A+C-2,A+C-6,B+C-10).CCK-8 cell proliferation and invasion was then tested in control group and the experimental group.Results The sgRNA was successfully linked into PX-330 plasmid.Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out,and the knockdown efficiency was above 70％,but the total amount of fibmnectin did not change significantly.Three monoclones of EDA absent SACC-83 cells were successfully selected with diminished migration and proliferation.Conclusions The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.
