| Noggin基因沉默表达载体的构建及效果评价 |
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| 引用本文： | 马雨楠,游颖,孙兆增,曾林.Noggin基因沉默表达载体的构建及效果评价[J].实验动物与比较医学,2016,24(1). |
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| 作者姓名： | 马雨楠 游颖 孙兆增 曾林 |
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| 作者单位： | 军事医学科学院实验动物中心,军事医学科学院实验动物中心,军事医学科学院实验动物中心,军事医学科学院实验动物中心 |
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| 基金项目： | 国家自然科学基金项目（面上项目，重点项目，重大项目）;国家科技支撑计划 |
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| 摘 要： | 目的构建慢病毒介导的Noggin RNAi干扰序列，并分析这些干扰序列对Noggin基因的沉默效果。方法针对目的基因Noggin的mRNA设计四条干扰序列，并将这些序列连接到Lenti-KD慢病毒载体，将重组质粒瞬时转染HEK-293T包装细胞，获得重组慢病毒。将重组病毒感染MC3T3-E1细胞，利用Puromycin进行筛选，获得稳定表达细胞系。通过实时荧光定量PCR和Western Blot技术分析不同干扰序列的干扰效果。结果实时荧光定量PCR结果显示，四种干扰序列对Noggin基因的表达都有一定的沉默效果，但只有shNoggin-1（P<0.01）对其表达影响显著。Western Blot结果显示，四种干扰序列中只有shNoggin-1（P<0.01）对Noggin的表达蛋白具有显著的降低作用。结论 获得了一种Noggin基因的干扰序列，该序列能够干扰Noggin基因mRNA的稳定性，从而影响蛋白的表达。该干扰序列可以用于部分敲除Noggin基因，从而用于研究Noggin基因的功能。
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| 关 键 词： | Noggin 基因沉默 表达载体 |
| 收稿时间： | 2015/7/14 0:00:00 |
| 修稿时间： | 2015/7/23 0:00:00 |
Construction and evaluation gene silencing vectors of Noggin |
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| Abstract: | Objective To construct the retroviral-mediated short hairpin RNA( shRNA) expression vectors of Noggin, and to analyze the silencing effect of the shRNA. Methods Four interference sequences were designed based on Noggin mRNA, and connected to Lenti-KD vectors. The recombinant plasmids were transiently transfected into HEK-293T cells, and finally obtained the recombinant virus. And then MC3T3-E1 cells were infected with the recombinant virus and screened by Puromycin. The gene silencing effect of shRNA was evaluated using RT-PCR and Western Blot. Results RT-PCR results showed that the four interference sequences all had silencing effect on the expression of Noggin, but only shNoggin-1( P<0.01) was differed significantly. Western Blot result showed that among the four interference sequences, only shNoggin-1( P<0.01) had a significantly decreased effect on the expression of Noggin. Conclusions One interference sequence of Noggin gene was obtained, which can interfere the stability of mRNA of the Noggin gene, there by affecting the expression of the protein. It is useful for the further study of unknown biological function of Noggin. |
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| Keywords: | Noggin Gene silencing Expression vector |
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