T细胞条件性敲除Spi1基因小鼠的繁育及鉴定  

Breeding and genotyping of T lymphocyte-conditional Spi1 knockout mice

作　　者：王卉卉 朱向玲 吴旭铭 张慧茹 周园园 王安琪 刘崇 涂佳杰 Wang Huihui;Zhu Xiangling;Wu Xuming;Zhang Huiru;Zhou Yuanyuan;Wang Anqi;Liu Chong;Tu Jiajie(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Education,Collaborative Innovation Center of Anti-inflammatory and Immune Medicines,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,抗炎免疫药物安徽协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2024年第4期595-599,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省高校杰出青年科研项目(编号:2022AH020052)。

摘　　要：目的繁育T细胞条件性敲除Spi1基因的小鼠并对其进行鉴定,为进一步探索Spi1编码蛋白PU.1的作用提供研究基础。方法将Lck-Cre小鼠与Spi1^(flox/flox)小鼠进行杂交繁育,通过聚合酶链式反应(PCR)和琼脂糖凝胶电泳鉴定小鼠基因型,筛选出基因型为Lck-Cre×Spi1^(flox/flox)的小鼠即为T细胞条件性敲除Spi1基因的纯合子小鼠。使用磁珠分选脾脏T淋巴细胞,并应用Western blot、实时荧光定量PCR(qPCR)及流式细胞术检测PU.1在T细胞中的敲除效率。结果Lck-Cre×Spi1^(flox/flox)小鼠基因稳定遗传。与Spi1^(flox/flox)小鼠相比,Lck-Cre×Spi1^(flox/flox)小鼠脾脏T细胞中的PU.1表达水平显著降低。结论该研究应用Cre/LoxP系统和CRISPR/Cas9技术成功构建了T细胞条件性敲除Spi1基因小鼠,为后续研究PU.1在T细胞相关疾病中的具体作用提供了可靠的动物模型。Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further investgation of the specific role of Spi1-encoded protein PU.1.Methods The Lck-Cre mice were mated with Spi1^(flox/flox)mice to obtain Lck-Cre×Spi1^(flox/flox)mice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR)and agarose gel electrophoresis.Magnetic beads were used to sort out the splenic T lymphocytes,and the knockdown efficiency of PU.1 in T cells was detected by Western blot,quantitative real-time PCR(qPCR)and flow cytometry.Results The Lck-Cre×Spi1^(flox/flox)mouse genotype was stably inherited.Compared with Spi1 flox/flox mice,the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1^(flox/flox)mice.Conclusion In this study,the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology,which provided a reliable animal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.

关 键 词：Spi1 CRE/LOXP系统 CRISPR/Cas9技术 T细胞 PU.1 条件性敲除 

分 类 号：R-332[医药卫生]