小鼠动力蛋白激活蛋白1-shRNA慢病毒载体的构建及在足细胞中的敲低效应
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上海市自然科学基金(09ZR1434800).


Construction of mouse dynactin-1-shRNA lentiviral vector and its knockdown efficiency in mouse podocytes
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Supported by National Natural Science Foundation of Shanghai (09ZR1434800).

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    摘要:

    目的 构建表达小鼠动力蛋白激活蛋白1(dynactin-1)特异性shRNA的慢病毒载体,并检测其对小鼠足细胞dynactin-1的敲低效果。方法 针对小鼠dynactin-1 mRNA序列,设计合成3种shRNA,克隆到入门质粒pENTR/pTER中,再利用LR反应重组到pLenti X2 Puro慢病毒目的 质粒,经过酶切测序鉴定后,将慢病毒质粒和包装质粒共转染293FT细胞,包装得到病毒颗粒。各组shRNA病毒载体转染小鼠足细胞后,用嘌呤霉素抗性筛选细胞,利用蛋白质印迹法检测各组细胞中dynactin-1蛋白的表达水平。结果 构建的各组shRNA入门质粒和慢病毒载体经酶切及测序鉴定正确;慢病毒载体与慢病毒包装质粒共转染293FT细胞,制备病毒颗粒,转染小鼠足细胞,经嘌呤霉素筛选获得稳定表达shRNA的足细胞系;蛋白质印迹检测结果 表明转染dynactin-1-shRNA组的dynactin-1蛋白表达降低。结论 构建的小鼠dynactin-1-shRNA慢病毒载体能有效降低小鼠足细胞中dynactin-1蛋白表达,为进一步研究dynactin-1在足细胞中的功能奠定了基础。

    Abstract:

    Objective To construct the lentiviral vector carrying shRNA of mouse dynactin-1 and to identify its knockdown efficiency of dynactin-1 by infecting mouse podocytes. MethodsThree pairs of shRNA targeting mouse dynactin-1 were synthesized and subcloned into BglⅡ-HindⅢ digested pENTR/pTER entry vectors. Then the entry vectors were transferred into pLenti X2 Puro destination vector by LR Clonase reaction. All the constructs were verified by restriction enzyme digestion and sequencing. The pLentiX2 Puro/dynactin-1-shRNA vectors and the packaging vectors were co-transfected into 293FT cells to produce dynactin-1-shRNA lentiviruses, which were used to transfect podocytes and the cells were screened by puromycin resistance. The expression of dynactin-1 protein in podcytes was analyzed by Western blotting analysis. ResultsRestriction enzyme digestion and sequencing analysis confirmed that the dynactin-1-shRNA lentivirus vector was successfully constructed. The podocyte cell lines stably expressing dynactin-1-shRNA were obtained by puromycin selection. Western blotting analysis indicated that dynactin-1 protein expression was down-regulated by dynactin-1-shRNA lentiviral vector in podocytes. ConclusionWe have successfully constructed lentiviral vector of dynactin-1-shRNA, which can effectively down-regulate dynactin-1 protein expression, providing a basis for further studying the role of dynactin-1 in podocytes.

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  • 收稿日期:2012-06-15
  • 最后修改日期:2012-06-27
  • 录用日期:2012-09-21
  • 在线发布日期: 2012-10-24
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