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

Abstract

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

CLC Number:

R-332

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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Construction and identification of Balb/c transgenic mice with NOD-SIRPA gene knocked in situ

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