Construction of a stable 4.1R gene knockout cell model in RAW264.7 cells using CRISPR/Cas9 technique
WANG Chengbo
KANG Qiaozhen
DING Cong
LI Yawen
LIANG Taotao
ZHANG Chenglong
WANG Wen
WANG Ting
Abstract：Objective To construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/Cas9 technique. Methods Three high-grade small-guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA-Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA-lentiCRISPRv2 plasmid,and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19-bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation. Conclusion We successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique,which may facilitate further investigation of the function of 4.1R in macrophages.
Keywords：CRISPR/Cas94.1Rgene knockoutRAW264.7 cellsmacrophages
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 1609-1614 )