组成型雄烷受体人源化小鼠模型的构建与验证  

Construction and validation of constitutive androstane receptor humanized mice model

作　　者：李海山[1] 谢文平[1] 周丽丽[1] 沈国林[1] 宋乃宁[1] 陈会明[1] LI Haishan;XIE Wenping;ZHOU Lili;SHEN Guolin;SONG Naining;CHEN Huiming(Institute of Chemicals Safety,Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院化学品安全研究所,北京100176

出　　处：《沈阳医学院学报》2022年第3期281-284,289,共5页Journal of Shenyang Medical College

基　　金：国家重点研发计划项目课题(No.2017YFF0211201);国家自然科学基金面上项目(No.81273125)。

摘　　要：目的:构建组成型雄烷受体(constitutive androstane receptor,CAR)人源化小鼠模型,进行活化特性有效性验证。方法:应用转录激活因子样效应物核酸酶技术构建CAR基因敲除小鼠模型;应用细菌人工染色体大片段序列转基因方法构建人源CAR全长序列转基因小鼠模型。对杂交后获得的CAR人源化小鼠模型进行测序鉴定和基因扩增片段鉴定,并分析人源CAR剪接体肝组织丰度特征。比较野生小鼠、CAR基因敲除小鼠、CAR人源化小鼠肝、肠组织CAR表达水平,采用荧光定量聚合酶链反应法检测给予物种特异性激活剂后肝组织靶基因细胞色素P4502B、3A相对表水平。结果:测序鉴定CAR基因敲除小鼠基因缺失10个碱基对序列,人源CAR序列整合进入小鼠基因组。CAR人源化小鼠剪接体肝组织丰度与人肝组织相同,对小鼠特异性激活剂无应答,给予人特异性激活剂后肝组织细胞色素P4502B、3A表达显著升高(P<0.01)。结论:构建的CAR人源化小鼠模型在肝组织生理表达水平、剪接体丰度和物种特异性激活剂应答方面均符合人源化特征。Objective:To construct the constitutive androstane receptor(CAR)humanized mice model and verify the effectiveness of activation characteristics.Methods:CAR gene knockout mice model was constructed by transcriptional activator-like effector nuclease technology.Human CAR full-length transgenic mice model was constructed by bacterial artificial chromosome.The CAR humanized mice model by hybridization were identified by gene amplification and sequencing.The abundance characteristics of human CAR splice variants in liver tissue were analyzed.The expression levels of CAR in liver and intestine tissues of wild type,CAR gene knockout,and CAR humanized mice were compared,and the relative expression levels of cytochrome P4502B and 3A in liver tissue after species-specific activator treatment were compared.Results:Sequencing identified that CAR gene knockout mice lacked 10 base pairs,and the human CAR sequence was integrated into the mouse genome.The abundance of splice variants in liver tissue of CAR humanized mice was the same as that of human liver tissue,and there was no response to mice specific activator.After human specific activator treatment,the expression of cytochrome P4502B and 3A in liver tissue of CAR humanized mice increased significantly(P<0.01).Conclusion:The constructed CAR humanized mice model accorded with humanized characteristics in terms of physiological expression level,splice variants abundance and species-specific activator response in liver tissue.

关 键 词：小鼠 组成型雄烷受体 基因编辑 验证 

分 类 号：R-332[医药卫生]