摘要
目的构建并鉴定胸腺上皮细胞(thymic epithelial cells,TECs)特异性敲除GSK-3β基因小鼠模型,评价其免疫学特征。方法采用小鼠胚胎干细胞(ES细胞,embryonic stem cells)打靶基因敲除技术,得到F0代的GSK-3β^(flox/flox)(GSK-3β^(f/f))小鼠;再采用Cre-LoxP系统进行小鼠的繁殖;PCR鉴定,筛选出基因型为GSK-3β^(f/f)FoxN1-Cre^(+/-)(GSK-3β^(-/-))小鼠,即为在TECs特异性敲除GSK-3β基因的小鼠。观察基因敲除鼠的一般生物学特征、繁殖能力和子代存活率。应用HE染色、流式细胞术及免疫荧光技术,比较基因敲除鼠和野生型小鼠免疫器官结构、胸腺、脾及外周血中免疫细胞比例及增殖能力差异。结果成功构建TECs特异性敲除GSK-3β基因小鼠模型。与野生型(wild type,WT)小鼠相比,GSK-3β^(-/-)小鼠一般生物学特征无明显差异,子代存活率>90%;衰老进程中GSK-3β^(-/-)小鼠胸腺比WT小鼠体积大,胸腺指数高,胸腺细胞数量多;脾中的初始T细胞及近期迁移细胞数量也显著多于WT小鼠。结论成功构建了TECs特异性敲除GSK-3β基因小鼠模型。为研究GSK-3β基因在TECs的功能和作用机制提供了很好的研究模型。
Objective To establish and characterize GSK-3βknockout thymic epithelial cells in miceand evaluate their immunological characteristics.Methods The Cre-LoxP system was used for the F0 generation of GSK-3β~(flox/flox)(GSK-3β~(f/f))mice with targeted gene knockout in embryonic stem cells.PCR was used to analyze GSK-3β~(f/f)FoxN1-Cre~(+/-)(GSK-3β~(-/-))mice with specific knockout of the GSK-3βgene in thymus epithelial cells.The general biological characteristics,reproductive ability,and progeny survival rate of gene knockout mice were observed.HE staining,flow cytometry,and immunofluorescence were used to compare the immune organ structure,thymus,spleen,and proportion and proliferation of immune cells in peripheral blood between knockout and wildtype mice.Results Mice with specific knockout of the GSK-3βgene in thymus epithelial cells were established successfully.Compared with wildtype mice,GSK-3β~(-/-)mice showed no significant difference in general biological characteristics,and the survival rate of offspring was 90%.In the aging process,the thymus of knockout mice was larger than that of wildtype mice with a higher thymus index and more thymic cells.The numbers of initial T cells and recently migrated cells in the spleen were significantly higher than those in wildtype mice.Conclusions Mice with specific knockout of the GSK-3βgene in thymic epithelial cells was established successfully.These mice provide a good tool to study the function and mechanism of the GSK-3βgene in thymus epithelial cells.
作者
李喜
曾艳
陈昌蓉
李论
余祥
周永芹
王德成
邓亚光
宋银宏
LI Xi;ZENG Yan;CHEN Changrong;LI Lun;YU Xiang;ZHOU Yongqin;WANG Decheng;DENG Yaguang;SONG Yinhong(Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,Three Gorges University,Institute of Infection and Inflammatory Injury,School of Basic Medicine,Three Gorges University,Yichang 443002,China;China Three Gorges Medical Laboratory in Hubei Province,Yichang 443000)
出处
《中国实验动物学报》
CAS
CSCD
北大核心
2023年第3期279-286,共8页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金项目(81671397,82102206)
湖北省卫健委重点项目(WJ2019H528)。
