[1]刘静静,刘秀盈,冯娅茹,等.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(01):10-15+99.[doi:10.3969/j.issn.1671-7414.2024.01.003]
　LIU Jingjing,LIU Xiuying,FENG Yaru,et al.Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology[J].Journal of Modern Laboratory Medicine,2024,39(01):10-15+99.[doi:10.3969/j.issn.1671-7414.2024.01.003]
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应用CRISPR/Cas9技术构建Raji-Luc CD19 KO淋巴瘤细胞系()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年01期
页码:
10-15+99
栏目:
论著
出版日期:
2024-01-15

文章信息/Info

Title:
Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology
文章编号:
1671-7414（2024）01-010-07
作者:
刘静静1; 刘秀盈1; 冯娅茹2; 冯义超1; 于梦圆1; 王建勋1; 3
（1. 北京中医药大学生命科学学院，北京 102488；2. 深圳细胞谷生物医药有限公司，广东深圳 518118；3. 深圳北京中医药大学研究院，广东深圳 518118）
Author(s):
LIU Jingjing1;  LIU Xiuying1;  FENG Yaru2;  FENG Yichao1;  YU Mengyuan1;  WANG Jianxun1; 3
（1.School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China; 2.Shenzhen Cell Valley Biopharmaceutical Co. Ltd, Guangdong Shenzhen 518118, China; 3.Shenzhen Research Institute, Beijing University of Chinese Medicine, Guangdong Shenzhen 518118, China）
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
分类号:
R392-33
DOI:
10.3969/j.issn.1671-7414.2024.01.003
文献标志码:
A
摘要:
目的　应用CRISPR/cas9 技术构建敲除CD19 的Raji-Luc 淋巴瘤细胞，并对其免疫逃逸能力进行初步验证。方法　构建PB-CRISPR-CD19 小向导RNA（small guide RNA, sgRNA）质粒，筛选最优的sgRNA 序列，用pCAG-PBase转座酶、PB-CD19 sgRNA 及PB-CRISPR-Cas9 共同电转染Raji-Luc 细胞，采用流式分选和极限稀释法筛选得到稳定敲除的单克隆细胞株。经过基因序列检测对敲除效果进行验证；酶标仪检测细胞系表面荧光素酶的表达情况；以实验室前期构建的CD19 CAR-T 和CD38 CAR-T 作为效应细胞，用荧光素酶化学发光法验证Raji-Luc CD19 KO 细胞株的免疫逃逸能力。结果　电转染制备的Raji-Luc CD19 KO 细胞转染效率较高，筛选所得两组单克隆细胞敲除效率均达到99% 以上，与原始Raji-Luc 细胞的荧光素酶表达无显著差异，且不能激活CD19 CAR-T 细胞对其进行杀伤。结论　成功构建了Raji-Luc CD19 KO 淋巴瘤细胞系。
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 PBCRISPR- 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.

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备注/Memo

备注/Memo:
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。
作者简介：刘静静（1999-），女，硕士研究生，主要从事CAR-T 细胞疗法方面研究，E-mail：ljj990821@163.com。
通讯作者： 王建勋（1973-），男，美籍，特聘教授，博士生导师，主要从事应用现代化基因与细胞治疗手段治疗血液疾病的临床转化研究，E-mail：Jianxun.Wang@bucm.edu.cn。
更新日期/Last Update: 2024-01-15