NOD-SIRPA基因原位敲入BALB/c转基因小鼠的构建及鉴定

doi:10.13481/j.1671-587X.20250301

摘要/Abstract

摘要：

目的基于成簇的规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）9技术构建NOD-信号调节蛋白α（SIRPA）基因敲入的小鼠模型，为构建更先进的人源化小鼠模型提供参考。方法基于CRISPR/Cas9技术，将NOD背景SIRPA基因敲入至BALB/c小鼠受精卵中，扩繁获得稳定基因型来源的纯合小鼠（SIRPA-KI鼠）进行实验，设计PCR引物，核酸电泳鉴定小鼠基因型。按照小鼠品系分为C57BL/6组、BALB/c组和SIRPA-KI组，各组随机取3只周龄相近小鼠进行实验。诱导成熟的骨髓巨噬细胞与人CD47-Fc融合蛋白共孵育，Streptavidin PE/Cy7染色，流式细胞术检测PE/Cy7平均荧光强度（MFI），比较各组小鼠SIRPA基因结合人CD47 Fc的能力。每只鼠尾静脉注射2×10⁹ 羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记的人红细胞，体内吞噬实验检测各组小鼠巨噬细胞体内吞噬人红细胞情况。随机取BALB/c和SIRPA-KI小鼠各1只诱导成熟骨髓巨噬细胞，体外吞噬实验检测各组小鼠巨噬细胞体内吞噬人红细胞情况。结果成功获得BLAB/c SIRPA^NOD/NOD纯合小鼠。流式细胞术，与C57BL/6组比较，BALB/c组小鼠MFI明显升高（P<0.01）；与C57BL/6组和BALB/c组比较，SIRPA-KI组小鼠MFI明显升高（P<0.01）。体内吞噬实验，C57BL/6组小鼠巨噬细胞整体清除人红细胞速率最快；巨噬细胞吞噬6 h时，与SIRPA-KI组比较，C57BL/6组人红细胞剩余百分率明显降低（P<0.01）且接近0%；与SIRPA-KI组比较，BALB/c组人红细胞剩余百分率明显降低（P<0.05）。体外吞噬实验，BALB/c组小鼠巨噬细胞明显吞噬人红细胞，且吞噬指数较高，为30.700%；与BALB/c组比较，SIRPA-KI组小鼠巨噬细胞吞噬指数明显降低（P<0.01）。结论通过CRISPR/Cas9技术向BALB/c品系敲入NOD背景SIRPA基因，成功构建对人CD47亲和力增强和可明显抑制吞噬人红细胞且具有优越的人类细胞异种移植效率的小鼠模型。

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

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 2×10? 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 SIRPA^NOD/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.

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

中图分类号:

R-332

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.

Mingyang TAO,Xin LI,Yanan LYU,Zheng HU. Construction and identification of Balb/c transgenic mice with NOD-SIRPA gene knocked in situ[J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 557-566.

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