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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作者姓名: | 傅鹏 原理 李金花 王春花 李林 原爱红 马骏 |
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作者单位: | 1. 同济大学附属同济医院肾内科,上海,200065 2. 上海市中西医结合医院急诊科,上海,200082 3. 江西省九江市中医医院肾六科,九江,332000 4. 第二军医大学长征医院肾内科,上海,200003 |
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基金项目: | 上海市自然科学基金(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.
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关 键 词: | 动力蛋白激活蛋白1 RNA干扰 慢病毒 足细胞 |
收稿时间: | 2012/6/15 0:00:00 |
修稿时间: | 2012/6/27 0:00:00 |
Construction of mouse dynactin-1-shRNA lentiviral vector and its knockdown efficiency in mouse podocytes |
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Authors: | FU Peng YUAN Li LI Jin-hu WANG Chun-hu LI Lin YUAN Ai-hong and MA Jun |
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Affiliation: | 1.Department of Nephrology,Tongji Hospital,Tongji University School of Medicine,Shanghai 200065,China 2.Department of Emergency Medicine,Shanghai TCM-integrated Hospital,Shanghai 200082,China 3.Department of Nephrology(6),Jiujiang TCM Hospital,Jiujiang 332000,Jiangxi,China 4.Department of Nephrology,Changzheng Hospital,Second Military Medical University,Shanghai 200003,China |
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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. Methods Three 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. Results Restriction 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. Conclusion We 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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Keywords: | dynactin 1 RNA interference lentivirus podocytes |
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