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夏一鸣,黄晓玲,仝慧慧,齐浩铭,莫丽冬,王 辰,范维佳,黄慧玲.中枢神经系统 TauT 基因敲除大鼠的构建及对线粒体 DNA 氧化损伤影响[J].中国比较医学杂志,2021,31(8):38~47.
中枢神经系统 TauT 基因敲除大鼠的构建及对线粒体 DNA 氧化损伤影响
Construction of TauT gene knockout in the central nervous system of rats and its effect on oxidative damage of mitochondrial DNA
投稿时间:2020-12-25  
DOI:10. 3969 / j.issn.1671-7856. 2021. 08. 006
中文关键词:  TauT 基因  条件敲除  CRISPR/ Cas9  Cre-loxP  大鼠  线粒体
英文关键词:TauT gene  conditional knockout  CRISPR/ Cas9  Cre-loxP  rat  mitochondria
基金项目:
作者单位E-mail
夏一鸣 天津医科大学,天津 300070 1565215506@ qq.com 
黄晓玲 天津医科大学,天津 300070  
仝慧慧 天津医科大学,天津 300070  
齐浩铭 天津医科大学,天津 300070  
莫丽冬 天津市环湖医院,天津市神经外科研究所,天津市脑血管与 神经变性重点实验室,天津 300350  
王 辰 天津市环湖医院,天津市神经外科研究所,天津市脑血管与 神经变性重点实验室,天津 300350  
范维佳 天津市环湖医院,天津市神经外科研究所,天津市脑血管与 神经变性重点实验室,天津 300350  
黄慧玲 天津市环湖医院,天津市神经外科研究所,天津市脑血管与 神经变性重点实验室,天津 300350 huanghuiling@ 126.com 
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中文摘要:
       目的 利用 CRISPR/ Cas9 和 Cre-loxP 技术相结合构建牛磺酸转运体( taurine transporter,TauT)在中枢神经系统中条件性敲除大鼠,并对其脑组织线粒体 mtDNA 及线粒体呼吸链酶活性进行初步研究。 方法 利用 CRISPR/ Cas9 和 Cre-loxP 技术获得 TauT 基因第 5 外显子两端含有 loxP 位点的杂合子大鼠(TauTloxP / WT )。 将获得的 TauTloxP / WT大鼠与 Nestin-Cre 大鼠交配,经繁殖和鉴定最终获得神经特异性 TauT 基因敲除大鼠(TauTloxP / loxP / Cre+ )。 通过 Real-time PCR、Westen blot、免疫组化对 TauTloxP / loxP / Cre+大鼠进行基因和蛋白表达检测,用 HE 染色观测其脑组织形态,并对脑组织 mtDNA 拷贝数和线粒体呼吸链酶(Ⅰ/ Ⅱ/ Ⅲ/ Ⅳ/ Ⅴ)活性进行检测。 结果 与野生型大鼠相 比,TauTloxP / loxP / Cre+大鼠脑组织中 TauT 基因和蛋白表达显著降低,TauT 基因在中枢神经系统被成功敲除,模型构建成功;HE 染色显示 TauTloxP / loxP / Cre+大鼠脑细胞数量和密度有所降低,而老年 TauTloxP / loxP / Cre+大鼠脑中细胞病变更加明显。 此外,与野生型大鼠相比,TauTloxP / loxP / Cre+大鼠脑组织线粒体呼吸链复合酶Ⅰ、Ⅲ、Ⅳ、Ⅴ酶活性显著降低,但 mtDNA 拷贝数却明显上升。 结论 利用 CRISPR/ Cas9 和 Cre-loxP 技术成功了构建中枢神经系统 TauT 基因敲除大鼠模型,并初步验证了此模型对脑组织及其线粒体呼吸链酶和 mtDNA 的影响,为研究牛磺酸及 TauT 对脑组织的分子机制提供了新的模型平台。
英文摘要:
       Objective CRISPR/ Cas9 and Cre-loxP were used to construct the conditional knockout of taurine transporter (TauT) in the central nervous system of rats. The mitochondrial mtDNA and mitochondrial respiratory chain enzyme activities in brain tissues were studied. Methods CRISPR/ Cas9 and Cre-loxP techniques were used to obtain heterozygous rats (TauTloxP / WT ) with loxP at both ends of exon 5 of the TauT gene. The obtained TauTloxP / WT rats were mated with Nestin-Cre rats. After breeding and identification, neural-specific TauT gene knockout rats ( TauTloxP / loxP / Cre+ ) were obtained. The gene and protein expressions of TauTloxP / loxP / Cre+ rats were detected by Real-time PCR, Western blot, and immunohistochemistry. The morphology of brain tissue was observed by hematoxylin-eosin staining. The mtDNA copy number and mitochondrial respiratory chain enzyme ( I/ II/ III/ IV/ V) activity in brain tissue were examined. Results Compared with wild-type rats, TauT gene and protein expressions in TauTloxP / loxP / Cre+ rat brain tissues were significantly decreased, and the TauT gene was successfully knocked out in the central nervous system indicating the knockout model was successfully constructed. Hematoxylin-eosin staining showed that the number and density of brain cells were decreased in TauTloxP / loxP / Cre+ rats and cytopathic changes more obvious in the brains of old TauTloxP / loxP / Cre+ rats. In addition, compared with wild-type rats, the activities of mitochondrial respiratory chain complex enzymes Ⅰ, Ⅲ, Ⅳ, and Ⅴ were significantly decreased, but the copy number of mtDNA was significantly increased. Conclusions The model of TauT gene knockout in the central nervous system of rats was successfully constructed using CRISPR/ Cas9 and Cre-loxP technology. The effects of TauT knockout in the central nervous system on brain tissues, respiratory chain enzymes, and mtDNA of mitochondria were verified, providing a new model platform for the study of the molecular mechanisms of taurine and TauT in brain tissues.
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