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| 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 |
| 傅鹏1△,原理2△,李金花3,王春花1,李林4,原爱红1*,马骏1* |
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(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
△共同第一作者
*通信作者) |
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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。 |
| 关键词: 动力蛋白激活蛋白1 RNA干扰 慢病毒 足细胞 |
| DOI：10.3724/SP.J.1008.2012.001077 |
| 投稿时间：2012-06-15修订日期：2012-06-27 |
| 基金项目:上海市自然科学基金(09ZR1434800). |
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| Construction of mouse dynactin-1-shRNA lentiviral vector and its knockdown efficiency in mouse podocytes |
| FU Peng1△,YUAN Li2△,LI Jin-hua3,WANG Chun-hua1,LI Lin4,YUAN Ai-hong1*,MA Jun1* |
(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
△Co-first authors.
*Corresponding authors.) |
| 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. |
| Key words: dynactin 1 RNA interference lentivirus podocytes |
