现代检验医学杂志2024，Vol.39Issue(1)：10-15,99,7.DOI:10.3969/j.issn.1671-7414.2024.01.003
应用CRISPR/Cas9技术构建Raji-Luc CD19 KO淋巴瘤细胞系
Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology
摘要
Abstract
Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.关键词
嵌合抗原受体-T细胞/白细胞分化抗原19 KO/电转染/淋巴瘤/Raji-Luc细胞Key words
CAR-T/CD19 KO/electro transfection/lymphoma/CRISPR/Cas9分类
医药卫生引用本文复制引用
刘静静,刘秀盈,冯娅茹,冯义超,于梦圆,王建勋..应用CRISPR/Cas9技术构建Raji-Luc CD19 KO淋巴瘤细胞系[J].现代检验医学杂志,2024,39(1):10-15,99,7.基金项目
北京中医药大学高层次人才科研启动经费项目(9011451310032):中医药调节肿瘤免疫的生物学机制研究. （9011451310032）
