摘要
目的应用CRISPR/Cas9技术构建肿瘤坏死因子受体相关因子2(TRAF2)杂合子敲除小鼠,用于研究TNF-α-TRAF2信号转导异常介导的炎症免疫性疾病的病理机制及新靶点药物的开发。方法构建靶向敲除TRAF2基因的载体,通过显微注射,将先导RNA和Cas9 mRNA导入到C57BL/6JGpt小鼠的受精卵中,介导小鼠TRAF2基因突变,提取鼠尾蛋白,通过PCR和Western blot测定F0代基因型,成功获得TRAF2^(+/-)小鼠。使用F0代小鼠与C57BL/6JGpt野生型小鼠回交,获得稳定的TRAF2^(+/-)小鼠用于扩繁及后续实验。检测TRAF2^(+/-)小鼠体质量;Western blot检测TRAF2^(+/-)小鼠脾脏、肝脏及肾脏组织中TRAF2表达;HE染色检测TRAF2^(+/-)小鼠脾脏、肝脏及肾脏组织的发育情况。结果利用引物进行PCR鉴定,结果表明TRAF2^(+/-)小鼠在679 bp出现目的条带;Western blot结果表明,与野生型组比较,TRAF2^(+/-)小鼠鼠尾蛋白中TRAF2的表达明显下降(P<0.01)。与野生型小鼠比较,TRAF2^(+/-)小鼠体质量减轻(P<0.05);Western blot结果表明,与野生型小鼠比较,TRAF2^(+/-)小鼠的脾脏、肝脏及肾脏组织中TRAF2蛋白表达均降低(P<0.01)。HE染色结果表明,与野生型小鼠比较,TRAF2^(+/-)小鼠脾脏、肝脏及肾脏组织内细胞形态无明显差异。结论成功构建TRAF2^(+/-)小鼠,为探究TRAF2在发育调控中的作用、揭示TNF-α-TRAF2信号异常介导的炎症免疫疾病机制以及筛选相关药物靶点提供了重要动物模型。
Objective To generate heterozygous TRAF2 knockout mice,the CRISPR/Cas9 technology was successfully employed.These mice were served as a valuable model to explore the pathological mechanisms underlying inflammatory and immune disorders mediated by abnormal TNF-α-TRAF2 signaling and to develop new therapeutic targets.Methods A vector targeting the knockout of the TRAF2 gene was constructed.Lead RNA and Cas9 Mrna were introduced into the fertilized eggs of C57BL/6JGpt mice through microinjection to mediate the TRAF2 gene mutation in mice.The mouse tail protein was extracted and the genotype of the F0 generation was determined by PCR and Western blot.TRAF2^(+/-)mice were successfully obtained.F0 generation mice were backcrossed with C57BL/6JGpt wild-type mice to obtain stable TRAF2^(+/-)mice for propagation and subsequent experiments.The body weight of TRAF2^(+/-)mice was detected;Western blot was used to detect the expression of TRAF2 in the spleen,liver and kidney tissues of TRAF2^(+/-)mice.The development of spleen,liver and kidney tissues in TRAF2^(+/-)mice was detected by HE staining.Results PCR identification using specific primers demonstrated that TRAF2^(+/-)mice exhibited a target band at 679 bp.Western blot analysis results indicated that,compared with the WT group,the expression of TRAF2 in the tail protein of TRAF2^(+/-)mice was significantly reduced(P<0.01).TRAF2^(+/-)mice had a lower body weight compared to their littermate WT mice(P<0.05).Western blot analysis showed that,relative to the WT group,the expression of TRAF2 protein in the spleen,liver,and kidney tissues of TRAF2^(+/-)mice was decreased(P<0.01).HE staining results indicated that there were no significant differences in cellular morphology in the spleen,liver,and kidney tissues between TRAF2^(+/-)mice and WT mice.Conclusion The successful construction of TRAF2^(+/-)mice has provided an important animal model for exploring the role of TRAF2 in developmental regulation,revealing the mechanism of inflammatory immune diseases mediated by abnormal TNF-α-TRAF2 signaling,and screening related drug targets.
作者
王伟康
左书俊
谷金涛
郭富媛
郭昊周
韩陈陈
魏伟
Wang Weikang;Zuo Shujun;Gu Jintao;Guo Fuyuan;Guo Haozhou;Han Chenchen;Wei Wei(Institute of Clinical Pharmacology,School of Pharmaceutical Sciences,Anhui Medical University,Key Laboratory of Anti Inflammatory and Immune Drugs of the Ministry of Education,Anhui Collaborative Innovation Center for Anti Inflammatory and Immune Drugs,Hefei 230032)
出处
《安徽医科大学学报》
北大核心
2025年第7期1291-1296,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:82104187、82173824)。
