Tianjin Medical Journal

Tianjin Med J ›› 2015, Vol. 43 ›› Issue (10): 1104-1107.doi: 10.11958/j.issn.0253-9896.2015.10.005

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Construction of Asxl2 gene knock out stable NIH3T3 cell line with CRISPR/Cas9n system

  

  1. 1 Department of Pharmacology,College of Basic Medical, Tianjin Medical University, Tianjin 300070, China；2 Community Health Service Center, Xiaobailou Street, Heping District；3 Deparment of Cell Biology,College of Basic Medical,Tianjin Medical University； 4 Tianjin Medical University Eye Hospital
  • Received:2015-01-30 Revised:2015-06-03 Published:2015-10-15 Online:2015-10-22

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Abstract:

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.

Key words: ASXL2, CRISPR/Cas9n, CRISPR system, epigenetic