基于Cre-LoxP系统的线粒体钙离子单向转运体脑条件性敲除小鼠模型的构建及鉴定  

Construction and identification of a mouse model with conditional knockout of mitochondrial Ca2+ uniporters in the mouse brain based on the Cre-LoxP system

作　　者：高俊 温开清 朱海珍 周涛[1] 李爱玲[1] 李腾[1] 张学敏[1] 潘欣[1] GAO Jun;WEN Kai-qing;ZHU Hai-zhen;ZHOU Tao;LI Ai-ling;LI Teng;ZHANG Xue-min;PAN Xin(National Center for Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《军事医学》2022年第7期518-523,共6页Military Medical Sciences

基　　金：国家杰出青年科学基金(82025028)。

摘　　要：目的 应用Cre-LoxP系统构建线粒体钙离子单向转运体(MCU)脑条件性敲除小鼠。方法 采用胚胎干细胞(ES细胞)打靶技术,将两个LoxP位点转入MCU基因5号外显子两端,构建MCU^(flox/+)小鼠。经与Nestin-cre工具小鼠交配获得MCU脑条件性敲除小鼠(MCU^(flox/+):Nestin-cre^(+))。采用鼠尾PCR进行基因型鉴定,Western印迹和实时荧光定量PCR(qPCR)技术分别从蛋白质和mRNA水平检测MCU的敲除效果。统计分析敲除小鼠生长发育和繁殖情况,旷场实验评价敲除小鼠自发活动性和适应性,高架O迷宫评价敲除小鼠焦虑情绪。结果 基因型鉴定、Western印迹和qPCR结果显示MCU脑条件性敲除小鼠成功构建,敲除小鼠繁殖能力正常,子代雌雄和基因型比例符合预期。与野生型小鼠相比,MCU脑条件性敲除小鼠体重明显降低,差异有统计学意义(P<0.05)。敲除小鼠在旷场中运动的总距离和运动时间明显高于野生型小鼠(P<0.05),在中间区域的停留时间、高架O迷宫开放臂的进入次数和时间无显著性差异。结论 成功构建MCU脑条件性敲除小鼠,小鼠可正常生长繁育,发育略有迟缓,运动和适应能力正常,有高度活跃行为,无明显焦虑样情绪。Objective To construct a mouse model with conditional knockout of mitochondrial Cauniporters(MCUs)in the brain of mice using the Cre-LoxP technique. Methods MCUflox/+mice were constructed by transferring two LoxP loci to both ends of exon 5 of MCU genes using the embryonic stem(ES)cell-based gene targeting technique. Mice with conditional knockout of MCUs in the brain(MCU^(flox/flox):Nestin-cre^(+))were obtained by mating with Nestin-cre+tool mice by MCUflox/flox. Genotyping of progeny was determined by PCR analysis on mouse-tail DNA. Expressions of MCU protein and mRNA were detected by Western blotting and quantitative real-time PCR(qPCR),respectively. The growth,development and reproduction of knockout mice were statistically analyzed. The spontaneous activity and adaptability of knockout mice were monitored by an open field test. Mice were tested for anxiety behavior on the elevated O-maze. Results A mouse model with conditional knockout of MUCs in the brain was established after being confirmed by genotype identification,Western blotting and qPCR. The reproductive capacity of the knockout mice was normal,and the proportion of male and female offsprings and genotypes met expectations. Compared to wild-type mice,the body mass of mice with conditional knockout of MCUs in the brain was considerably increased,and the difference was statistically significant(P<0.05).The total distance and movement time of the knockout mice in the open field were significantly longer than those of the wild-type mice(P<0.05). There was no significant difference in the stay time in the center of the open field or in the number of times rats entered the open arm of the elevated O-maze between knockout and wild-type mice. Conclusion Mice with conditional knockout of MUCs in the brain have been successfully constructed,which are able to grow and reproduce normally,with slightly delayed development. The mice have normal motor skills and adaptive abilities,highly active behavior,and no obvious anxiety.

关 键 词：Cre-LoxP系统 线粒体钙离子单向转运体 基因敲除 脑 小鼠 胚胎干细胞 聚合酶链反应 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]