Construction of Asxl2 gene knock out stable NIH3T3 cell line with CRISPR/Cas9n system
10.11958/j.issn.0253-9896.2015.10.005
- VernacularTitle:利用CRISPR/Cas9n系统构建Asxl2基因敲除的NIH3T3稳定细胞系
- Author:
Jiaping FANG
;
Xiujuan ZHAO
;
Yan QI
;
Xi WANG
;
Xudong WU
;
Jianshi LOU
- Publication Type:Journal Article
- Keywords:
ASXL2;
CRISPR/Cas9n;
CRISPR system;
epigenetic
- From:
Tianjin Medical Journal
2015;(10):1104-1107
- CountryChina
- Language:Chinese
-
Abstract:
Objective To knock out Asxl2 gene in murine embryonic fibroblast cell line NIH3T3 using CRISPR/Cas9n system. Methods A pair of sgRNAs which targeted exon 5 of Asxl2 gene were designed and subcloned into the pX462 vec?tor. The recombined plasmids were verified by sequencing and transfected into NIH3T3 cell line. Single cells were isolated through serial dilutions, followed by an expansion period to obtain new monoclonal cell lines. The genomic DNA of the new monoclonal cell lines was extracted and a DNA fragment flanked the target site was amplified by genotyping PCR then se?quenced. Lastly, western blotting were applied to confirm whether Asxl2 was successfully knocked out. Results The CRIS?PR/Cas9n plasmids that targeted Asxl2 were successfully constructed. NIH3T3 cells were co-transfected with the two recom?binant constructs. After puromycin selection, subclonal cell lines were obtained and one of them was validated by genotyping PCR-sequencing. Western blotting also confirmed that Asxl2 was completely depleted in the NIH3T3 cell line. Conclu?sion CRISPR/Cas9n plasmids that targeted Asxl2 were successfully constructed therefore a Asxl2 knockout NIH3T3 stable cell line was established via this system.
