| 小鼠CDK4基因RNAi慢病毒载体的构建及体外研究 |
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| 引用本文： | 降风,王雪贞,卜碧涛,张苏明,张旻.小鼠CDK4基因RNAi慢病毒载体的构建及体外研究[J].国外医学：物理医学与康复学分册,2008,3(6). |
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| 作者姓名： | 降风 王雪贞 卜碧涛 张苏明 张旻 |
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| 作者单位： | [1]华中科技大学同济医学院附属同济医院神经内科,武汉430030 [2]内蒙古自治区医院神经内科,呼和浩特010017 |
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| 基金项目： | 国家自然科学基金
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| 摘 要： | 目的:探讨CDK4在神经系统变性疾病中的作用,并构建携带小鼠CDK4-siRNA的慢病毒载体进行体外研究。方法:设计3个CDK4-siRNA序列和1个无关对照序列,化学合成后退火形成双链,克隆到酶切的pSIH1-H1-copGFP siRNA载体。重组质粒经PCR和测序鉴定后,脂质体介导下转染BV-2细胞,Western blot检测CDK4的表达,筛选出干扰效果最佳的pSIH1-siRNA2。将对照序列和pSIH-siRNA2分别与慢病毒包装质粒共转染293T细胞生成病毒,收集病毒上清并浓缩,测定浓度。结果:慢病毒感染BV-2细胞后可表达GFP,RT-PCR和Western blot检测CDK4的表达明显下降。结论:成功构建携带CDK4-siRNA的慢病毒载体,体外研究显示其能明显下调CDK4的表达。
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| 关 键 词： | CDK4 神经系统变性疾病 RNA干扰 慢病毒载体 |
Construction and Identification of Lentivector-mediated CDK4-RNAi |
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| JIANG Feng,WANG Xue-zhen,BU Bi-tao,ZHANG Su-ming,ZHANG Min.Construction and Identification of Lentivector-mediated CDK4-RNAi[J].Neural Injury and Functional Reconstruction,2008,3(6). |
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| Authors: | JIANG Feng WANG Xue-zhen BU Bi-tao ZHANG Su-ming ZHANG Min |
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| Institution: | JIANG Feng,WANG Xue-zhen,BU Bi-tao,ZHANG Su-ming,ZHANG Min.Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong University of Science , Technology,Wuhan 430030,China |
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| Abstract: | Objective:To study the role of CDK4 in pathogenesis of neurodegenerative disease.And to construct lentivector carrying murine CDK4-interference RNA(RNAi) for study in vitro.Methods:Three pairs of murine CDK4-siRNA and one pair of negative control sequence were designed,then cloned into pSIH1-H1-copGFP siRNA vector.The above recombinants were transfected into BV-2 cells mediated by lipofectamine.The gene silencing efficacy of 3 targets was compared by western-blot analysis.The optimized pSIH1-siRNA2 and pSIH... |
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| Keywords: | CDK4 |
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