[1]王成博,康巧珍,丁聪,等.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,(12):1609.
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利用CRISPR/Cas9系统构建稳定敲除4.1R基因的RAW264.7细胞株()
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《南方医科大学学报》[ISSN:/CN:]

卷:
期数:
2017年12期
页码:
1609
栏目:
出版日期:
2017-12-20

文章信息/Info

Title:
Construction of a stable 4.1R gene knockout cell model in RAW264.7 cells using CRISPR/ Cas9 technique
作者:
王成博康巧珍丁聪李雅雯梁桃桃张成龙王文王婷
关键词:
CRISPR/Cas9系统4.1R基因敲除RAW264.7细胞
Keywords:
CRISPR/Cas9 4.1R gene knockout RAW264.7 cells macrophages
摘要:
目的利用CRISPR/Cas9系统构建稳定敲除4.1R基因的RAW264.7巨噬细胞株,为研究4.1R在巨噬细胞中的功能奠定基 础。方法根据CRISPR/Cas9 靶向原理设计并合成3 个特异性识别4.1R 基因的向导RNA(sgRNA),构建sgRNAlentiCRISPRv2 重组质粒并转入293T细胞中制备sgRNA-Cas9慢病毒,慢病毒侵染RAW264.7细胞,嘌呤霉素筛选出阳性细胞 并稀释至单克隆,Western blotting印记检测单克隆细胞中蛋白4.1R的表达,测序确认单克隆细胞中突变位点。结果Western blotting印迹检测结果表明筛选出的1株单克隆细胞中蛋白4.1R的表达完全缺失;测序结果表明该细胞株中4.1R基因发生了 19bp的缺失突变;并且4.1R基因敲除后,RAW264.7细胞的增殖能力显著增加。结论本研究利用CRISPR/Cas9系统成功的干 扰了巨噬细胞系RAW264.7细胞中4.1R的表达,为研究4.1R在巨噬细胞中的功能提供了有效工具。
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.
更新日期/Last Update: 1900-01-01