南方医科大学学报2017，Vol.37Issue(12)：1609-1614,6.DOI:10.3969/j.issn.1673-4254.2017.12.08
利用CRISPR/Cas9系统构建稳定敲除4.1R基因的RAW264.7细胞株
Construction of a stable 4.1R gene knockout cell model in RAW264.7 cells using CRISPR/Cas9 technique
摘要
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.关键词
CRISPR/Cas9系统/4.1R/基因敲除/RAW264.7细胞Key words
CRISPR/Cas9/4.1R/gene knockout/RAW264.7 cells/macrophages引用本文复制引用
王成博,康巧珍,丁聪,李雅雯,梁桃桃,张成龙,王文,王婷..利用CRISPR/Cas9系统构建稳定敲除4.1R基因的RAW264.7细胞株[J].南方医科大学学报,2017,37(12):1609-1614,6.基金项目
国家自然科学基金(81602537,81373119) （81602537,81373119）
河南省高等学校重点科研项目计划(17A180014)Supported by National Natural Science Foundation of China(81602537, 81373119). （17A180014）
