【摘要】 目的:制备线粒体缺陷的转基因小鼠,为研究线粒体相关疾病提供一个较好的动物模型。方法:将线粒体转录因子A(mitochondrial transcription factor A,TFAM)基因干扰慢病毒显微注射到小鼠受精卵中制备转基因小鼠。将转基因小鼠各组织制备冰冻切片观察荧光表达。用定量PCR(quantitative PCR,qPCR)和Western blot检测心脏TFAM基因的变化。用定量PCR检测心脏线粒体DNA拷贝数的变化。结果:首先构建了针对TFAM基因的干扰慢病毒载体,使用此病毒体外转染细胞后,能够有效的下调TFAM基因的转录和翻译。使用此病毒进行小鼠受精卵卵周注射,可以获得相应的转基因小鼠。病毒携带的绿色荧光蛋白在转基因小鼠的各个组织器官中均能很好的表达。转基因小鼠各组织TFAM转录水平明显降低。此转基因小鼠心脏中TFAM蛋白表达被有效的抑制,心肌细胞线粒体基因组拷贝数下降,心肌细胞线粒体呼吸链功能受损。结论:成功获得了线粒体转录因子A敲低的转基因小鼠。更多还原

【Abstract】 Objective: To produce mitochondria deficiency transgenic mice,providing a useful animal model for the investigation of mitochondria related diseases. Methods: Transgenic mice were produced by injection of mitochondrial transcription factor A( TFAM) RNAi lentivirus into zygotes. Frozen sections of tissues wre prepared to observe GFP expression. Variations of TFAM mRNA and protein were separately detected by quantitative PCR( qPCR) and western blot. Changes of mtDNA copy number were measured by qPCR. Results:We constructed lentiviral vectors expressing shRNA targeting TFAM,which markedly repressed the transcription and translation of TFAM in transduced MEF cells. TFAM knockdown mice were successfully produced by infection of the purified virus into the perivitelline space of single-cell embryo. Green fluorescence was observed in heart,liver,spleen,lung and kidney of transgenic mice. Using qPCR and western blot analysis,we found that the transcription of TFAM in the tissues of transgenic mice was significantly decreased. Moreover,reduced expression of TFAM in myocardium led to a decline of mitochondrial DNA( mtDNA) copy number,as well as an impaired respiratory chain function. Conclusion: A TFAM knockdown transgenic mice line was successfully constructed.更多还原