基于Cre-LoxP系统构建一种新的时间特异性突变NLRP3转基因小鼠模型

Construction of a novel time-specific NLRP3 transgenic mouse model by Cre-LoxP system

目的应用Cre-LoxP系统构建一种新的时间特异性NLRP3点突变转基因小鼠模型并对其进行鉴定。方法利用Cre-LoxP技术,构建NLRP3基因T346M点突变的flox杂合子(NLRP3^(flox)/+)小鼠。将该小鼠与广谱表达Cre重组酶工具鼠(CAG-Cre/ERT2,CreT)杂交繁育,获得基因型NLRP3^(flox)/+CreT^(+/-)小鼠,后者与NLRP3 T346M的flox纯合子(NLRP3^(flox)/flox)小鼠进一步交配获得NLRP3^(flox)/flox CreT^(+/-)(KI/KI)和NLRP3^(flox)/+CreT^(+/-)(KI/WT)小鼠,于6~8周时经他莫昔芬的诱导实现NLRP3 T346M突变基因的时间特异性敲入。小鼠繁育和鉴定过程中,用PCR方法检测鼠尾基因组DNA。采用Western blot法检测小鼠肝脏和心脏中NLRP3、pro-Caspase-1和Caspase-1的蛋白质表达水平。结果小鼠给予他莫昔芬后,NLRP3 T346M突变基因被诱导敲入。Western blot结果显示,KI/KI小鼠肝脏和心脏组织以及KI/WT小鼠心脏中NLRP3炎性小体效应蛋白Caspase-1的表达水平较同窝野生型小鼠显著上调。结论本研究基于Cre-LoxP技术成功构建了NLRP3 T346M突变转基因小鼠模型,该模型小鼠在他莫昔芬诱导后可出现自发性的NLRP3炎症小体活化,这为探究NLRP3炎症小体活化机制以及筛选和评价NLRP3炎症小体抑制剂提供了新的时间特异性的实验动物模型。

Aim To construct and identify a new time-specific NLRP3 point mutation transgenic mouse model by Cre-LoxP system.Methods Cre-LoxP system was used to generate NLRP3 T346M^(flox)heterozygous(NLRP3^(flox)/+)mice,which were then interbred with broad-spectrum expression Cre recombinase tool mice(CAG-Cre/ERT2,abbreviated as CreT)to produce NLRP3^(flox)/+CreT^(+/-)mice.In order to generate NLRP3^(flox)/flox CreT^(+/-)(KI/KI)and NLRP3^(flox)/+CreT^(+/-)(KI/WT)mice,NLRP3^(flox)/+CreT^(+/-)mice were crossed with NLRP3 T346M homozygous(NLRP3^(flox)/flox)mice.After that,time-specific NLRP3 T346M mutant gene knockin mice could be acquired by tamoxifen induction at 6~8 weeks old.PCR was used to detect the mouse tail genomic DNA during breeding and identification.Western blot was used to detect the protein expression levels of NLRP3,pro-Caspase-1 and Caspase-1 in liver and heart.Results After tamoxifen induction,PCR results confirmed that the NLRP3 T346M mutant gene was knocked in.Western blot analysis revealed that the expression levels of the NLRP3 inflammasome-effector protein Caspase-1 in liver and heart tissues of KI/KI mice and the heart of KI/WT mice were significantly up-regulated compared with wild-type littermates.Conclusions In this study,NLRP3 T346M mutant transgenic mouse model is successfully constructed based on Cre-LoxP technology,demonstrating the spontaneous activation of NLRP3 inflammasome after tamoxifen induction.Therefore,it could be a new time-specific experimental animal model for further exploration of the mechanism of NLRP3 inflammasome activation and for screening and evaluation of NLRP3 inflammasome inhibitors.