吉林大学学报（医学版）2025，Vol.51Issue(3)：557-566,10.DOI:10.13481/j.1671-587X.20250301
NOD-SIRPA基因原位敲入BALB/c转基因小鼠的构建及鉴定
Construction and identification of Balb/c transgenic mice with NOD-SIRPA gene knocked in situ
摘要
Abstract
Objective:To discuss the construction of a NOD-signal regulatory protein α(SIRPA)gene knock-in mouse model based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)9 technology,and to provide the reference for the establishment of more advanced humanized mouse models.Methods:Based on CRISPR/Cas9 technology,the NOD-background SIRPA gene was knocked into the BALB/c mouse fertilized eggs,and homozygous mice with stable genotypes(SIRPA-KI mice)were obtained through expansion and breeding for experiments;PCR primers were designed,and mouse genotypes were identified by nucleic acid electrophoresis.The mice were divided into C57BL/6 group,BALB/c group,and SIRPA-KI group according to their strains,and 3 mice with similar ages were randomly selected from each group for experiments.Mature bone marrow-derived macrophages were co-incubated with human CD47-Fc fusion protein,stained with Streptavidin PE/Cy7,and the mean fluorescence intensity(MFI)of PE/Cy7 was detected by flow cytometry to compare the ability of SIRPA in the mice from various groups to bind human CD47 Fc.Each mouse was intravenously injected with 2×10⁹ carboxyfluorescein diacetate,succinimidyl ester(CFSE)-labeled human red blood cells,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vivo phagocytosis assay.One BALB/c mouse and one SIRPA-KI mouse were randomly selected to induce mature bone marrow-derived macrophages,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vitro phagocytosis assay.Results:BLAB/c SIRPANOD/NOD homozygous mice were successfully obtained.The flow cytometry results showed that compared with C57BL/6 group,the MFI of the mice in BALB/c group was significantly increased(P<0.01);compared with C57BL/6 group and BALB/c group,the MFI of the mice in SIRPA-KI group was significantly increased(P<0.01).The in vivo phagocytosis assay results showed that the macrophages in C57BL/6 group exhibited the fastest overall clearance rate of human red blood cells;at 6 h of macrophage phagocytosis,compared with SIRPA-KI group,the residual percentage of the human red blood cells in C57BL/6 group was significantly decreased(P<0.01)and was closed to 0%;compared with SIRPA-KI group,the residual percentage of the human red blood cells in BALB/c group was significantly decreased(P<0.05).The in vitro phagocytosis assay results showed that the macrophages in BALB/c group significantly phagocytosed the human red blood cells,with a high phagocytic index of 30.7%;compared with BALB/c group,the phagocytic index of the macrophages in SIRPA-KI group was significantly decreased(P<0.01).Conclusion:The study successfully constructs a mouse model with enhanced affinity for human CD47 and significantly inhibited phagocytosis of human red blood cells by knocking the NOD-background SIRPA gene into the BALB/c strain using CRISPR/Cas9 technology,providing a superior human cell xenotransplantation efficiency.关键词
信号调节蛋白α/BALB/c小鼠/C57BL/6小鼠/基于成簇的规则间隔短回文重复序列/基于成簇的规则间隔短回文重复序列相关蛋白9技术Key words
Signal regulatory protein α/BALB/c mice/C57BL/6 mice/Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 technology分类
医药卫生引用本文复制引用
陶明阳,李馨,吕亚楠,胡正..NOD-SIRPA基因原位敲入BALB/c转基因小鼠的构建及鉴定[J].吉林大学学报（医学版）,2025,51(3):557-566,10.基金项目
国家自然科学基金重点专项(82241224) （82241224）
