eNOS基因敲除大鼠模型的建立及表型初步研究  

Establishment of eNOS Knockout Rat Model and Phenotypic Study

作　　者：杨葳[1] 周生来[1] 董婉维[1] 王惟[1] 于洋[1] 郭晓冲[1] 张婀娜[1] 王宏宇 郑志红[1] 

机构地区：[1]中国医科大学实验动物部,沈阳110001 [2]南京徇齐生物技术有限公司,南京210061

出　　处：《实验动物与比较医学》2015年第4期271-276,共6页Laboratory Animal and Comparative Medicine

基　　金：国家科技重大项目（2011ZX09307-302-02）,辽宁省科技计划项目（2012408002）

摘　　要：目的建立内皮型一氧化氮合酶（eNOS）基因敲除大鼠模型。方法利用锌指核酸酶（ZFN）技术，通过显微注射的方法将编码eNOS特异性锌指酶的mRNA注入SD大鼠受精卵并移植到同期受孕的SD受体母鼠输卵管内。出生后仔鼠用PCR产物测序的方法鉴定基因型。对敲除大鼠进行外观观察，HE染色分析组织结构形态。通过Westernblot分析敲除大鼠模型的心脏和肾脏组织中eNOS的表达情况。测量8～16周大鼠体质量，分析eNOS基因敲除对体质量的影响。采用尾套法测量大鼠心率、血压、收缩压和舒张压，比较与野生型大鼠的差异。结果通过显微注射方法共注射441枚受精卵，存活365枚，分别移入12只SD大鼠输卵管，共产出23只F0代大鼠。PCR产物测序分析获得4只阳性原代大鼠，对8号大鼠筛选获得纯合子。阳性纯合子大鼠表现为肢体缺损，HE染色显示动脉血管结构形态异常。Westernblot结果显示，eNOS敲除大鼠的心脏和肾脏组织中不表达eNOS蛋白。敲除大鼠的体质量明显低于野生型大鼠，体质量增长幅度慢。血压、收缩压、舒张压均高于野生型SD大鼠，有极显著差异（P〈0．01）。eNOS敲除雌鼠心率低于野生型SD雌鼠（P〈0．05）。eNOS敲除雄鼠心率与野生型SD雄鼠相比无统计学差异。结论成功建立eNOS基因敲除大鼠模型，该模型或可成为高血压疾病研究的理想动物模型。Objective To establish an endothelial nitric oxide synthase （eNOS） knockout rat model. Methods The encoding eNOS specific Zinc-finger nucleases （ZFN） mRNA was transcripted in vitro and purified, then injected into zygotes. The injected zygotes were transferred into oviducts of pseudopreg- nant rats. The genotype of knockout rats were identified by PCR and sequencing. The histopathological changes were observed by HE staining. The eNOS expression were detected by Western blot. The body weight differences were detected at 8-16 weeks ages, and the blood pressures, systokic pressures, diastolic pressures and heart rates were determined at 8 weeks age by using a tail-cuff system. Result Total of 441 zygotes were injected and 365 zygote ceils were transplanted into oviducts of 12 recipients. Twenty-three offspring were born, and the results of PCR and sequencing showed that 4 offsprings were eNOS knockout rats. The screening result showed No.8 offspring was homozygous, and it exhibited limb defect, abnormal structures of artery. The results of Western blot showed that eNOS were not expressed in the kidney and heart in eNOS knockout rats. The comparison of body weights showed that the effect of eNOS genotype were significant different. Blood pressures, systokic pressures and diastolic pressures were higher in eNOS knockout rats than the SD rats, and there were significant differents. Heart rates were lower in the females eNOS knockout rats, but in the males eNOS knockout rats there were not significantly different. Conclusion eNOS knockout rats have been established＇successfully. This rat model will contribute to research of hypertension.

关 键 词：内皮型一氧化氮合酶(eNOS) 锌指核酸酶(ZFN) 大鼠 基因敲除 高血压 

分 类 号：Q95-33[生物学—动物学]