通过CRISPR/Cas9技术构建Apoe基因敲除小鼠模型及其表型研究  

Use of CRISPR/Cas9 system for establishment and characterization of Apoe gene knockout mice model

作　　者：柯璐 曹愿 谷文达[1] 刘甦苏[1] 孙晓炜 赵皓阳 翟世杰 郭怀勇 娄玥 范昌发[1] KE Lu;CAO Yuan;GU Wenda;LIU Susu;SUN Xiaowei;ZHAO Haoyang;ZHAI Shijie;GUO Huaiyong;LOU Yue;FAN Changfa(Institute for Laboratory Animal Resources,National Institutes for Food and Drug Control(NIFDC),Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院实验动物资源研究所,北京102629

出　　处：《中国实验动物学报》2024年第11期1390-1398,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点研发计划(2023YFC3402000)。

摘　　要：目的本研究通过CRISPR/Cas9技术构建Apoe基因敲除小鼠模型,以进一步探讨Apoe在血脂代谢和形成动脉粥样硬化病症中的功能。方法针对C57BL/6J小鼠Apoe基因设计2个sgRNA靶位点,并将sgRNA和Cas9 mRNA共同注入受精卵,移植至ICR受体鼠,获得F0代小鼠,通过提取鼠尾DNA以及PCR筛选,获得阳性小鼠。通过荧光定量逆转录PCR法检测小鼠各组织中Apoe mRNA表达水平。检测小鼠血清中的血脂指标,并通过油红O染色主动脉血管,以检测动脉内膜脂质的积聚。结果PCR及测序结果确认成功构建了Apoe基因敲除小鼠(C57BL/6-Apoe^(em1)/Nifdc,简称Apoe KO小鼠);实时荧光定量逆转录PCR实验结果显示,Apoe KO纯合小鼠(Apoe^(-/-))的肝、大脑、脾、肾和肺组织中的Apoe mRNA显著降低;血清总胆固醇、低密度脂蛋白胆固醇水平显著升高,且雄性小鼠的血清高密度脂蛋白胆固醇水平显著降低;主动脉血管油红O染色显示,在相同且食用正常饲料条件下,野生型(wild-tpye,WT)小鼠无脂质堆积,Apoe^(-/-)小鼠则呈现大量红色脂质斑块。结论本研究成功构建Apoe基因敲除小鼠,该纯合子小鼠在非高脂饲料下呈典型的异常血脂代谢及主动脉内膜脂质积聚特征,为Apoe KO小鼠资源提供了背景数据,为血脂代谢异常表型研究提供新模型。Objective The CRISPR/Cas9 system was utilized to generate an Apoe knockout mice model to support further investigations of the role of Apoe in lipid metabolism and atherosclerosis.Methods Two single guide RNAs designed for Apoe in C57BL/6J mice were co-injected with Cas9 mRNA into fertilized eggs,followed by transplantation into ICR recipient mice to obtain F0 generation mice.KO mice were identified by polymerase chain reaction(PCR)screening of tail DNA.Apoe mRNA expression in various tissues was assessed by quantitative real-time PCR and lipid indexes were measured in serum samples.Lipid accumulation in the inner lining of aortic vessels was detected by oil red O staining.Results PCR and sequencing confirmed the successful construction of Apoe KO mice(C57BL/6-Apoe^(em1)/Nifdc).Apoe mRNA levels were significantly reduced in the liver,brain,spleen,kidney,and lung tissues of Apoe KO homozygous mice(Apoe^(-/-)),as shown by reverse transcription quantitative real-time PCR.Serum total cholesterol and low-density lipoprotein cholesterol levels were increased in Apoe^(-/-)mice,and high-density lipoprotein cholesterol levels were decreased in male Apoe^(-/-)mice.Extensive lipid plaques were observed in the inner lining of the arteries in Apoe^(-/-)mice compared with WT mice,under normal chow consumption conditions.Conclusions This study successfully established an Apoe KO mice model exhibiting a typical abnormal lipid metabolism phenotype with arterial lipid accumulation,even without a highfat diet intervention.This work provides background data for the Apoe KO mice resource and a new model for the study of abnormal lipid metabolism.

关 键 词：APOE C57BL/6-Apoe^(em1)/Nifdc CRISPR/Cas9 血脂代谢 主动脉 

分 类 号：Q95-33[生物学—动物学]