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RNA干扰:一种快速关闭基因的途径 总被引:1,自引:0,他引:1
RNA干扰是双链RNA分子在mRNA水平关闭相应序列基因表达的过程,发现于植物、线虫。最近在哺乳动物细胞中也获得了满意的结果,这对研究哺乳动物,尤其是人类基因的功能将提供帮助。 相似文献
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RNAi是一种高度特异化的mRNA降解过程,能在哺乳动物细胞中诱导特异的基因沉默。这一发现为人类某些疾病的治疗研究开辟了新的途径。随着研究的不断深入,RNA i在抗病毒研究中受到越来越多的关注。本文拟就RNA i的机制、siRNA靶系列的选择及制备和抗病毒研究现状作一综述。 相似文献
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RNA干扰广泛存在于真核细胞中, 其作用机制已得到比较清楚阐明.虽然在原核细胞中已发现微小RNA(miRNA)参与的基因沉默调节细菌基因表达, 但尚未发现小干扰RNA (siRNA)参与的基因沉默机制.近年在原核细胞基因组中发现一类新的成簇的规律间隔的短回文重复序列 (clustered regularly interspaced short palindromic repeat, CRISPR)和与其相关的基因.生物信息学分析和预测表明两者构成的CRISPR相关系统介导原核细胞RNA干扰, 进而发挥免疫防御功能.这一假说新近得到了实验证实, 与此同时首次揭示了原核细胞的适应性免疫机制.这种防御机制不同于真核细胞适应性免疫机制, 除具特异性外, 还具有遗传性. 相似文献
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用 RNA 干扰 (RNA interference , RNAi) 技术抑制哺乳动物细胞中外源报告基因的表达,以探讨该过程中 RNAi 作用的剂量和时间效应 . 应用 Lipofectamine 2000 将外源报告基因的表达载体与编码短发夹 RNA (short hairpin RNA , shRNA) 的质粒共转染 HEK293H 细胞,观察 shRNA 载体对报告基因的抑制效应 . 转染后, shRNAs 的瞬时表达可特异地抑制细胞内报告基因的表达 . 在共转染后 12 , 24 , 48 , 60 , 72 , 96 h 时检测 EGFP (enhanced green fluorescent protein , EGFP) 基因 mRNA 及蛋白质表达水平,结果显示, EGFP mRNA 及蛋白质表达在 12 h 时略有降低, 24~48 h 时表达逐渐降低, 48~72 h 时降低最明显,其后 EGFP 表达水平逐渐恢复 . 提示该过程中 RNAi 效应呈现由弱到强、又由强到弱的逐渐消逝趋势 . 共转染一系列剂量比例的 EGFP 干扰载体与靶载体的结果表明,在一定剂量范围内, RNA 干扰载体所介导的抑制效应与干扰载体剂量大小有关,当其剂量进一步加大足以抑制外源基因表达时,抑制效应则维持在一“平台期” . 此外,通过 RNAi 抑制 HeLa 细胞、 HEK293 细胞中荧光素酶基因的表达, 荧光素酶活性变化也表现出上述类似的效应 . 这些结果表明,在体外哺乳动物细胞中,基于表达载体的 RNAi 作用呈现剂量和时间依赖性效应 . 这为基于载体表达的 RNAi 技术应用研究提供了一定的理论参考及依据 . 相似文献
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Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis
Marco Lezzerini Koen van de Ven Martijn Veerman Stanley Brul Yelena V. Budovskaya 《PloS one》2015,10(4)
In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded RNA (dsRNA) to knockdown translation of targeted mRNAs. However, it has been shown that E. coli is mildly pathogenic to C. elegans and this pathogenicity might influence aging and the accuracy of the RNAi-screening during aging may as well be affected. Here, we describe a novel system that utilizes the non-pathogenic bacterium Bacillus subtilis, to express dsRNA and therefore eliminates the effects of bacterial pathogenicity from the genetic analysis of aging. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(4):776-781
Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species. 相似文献
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U6和H1双启动子载体用于RNAi的实验研究 总被引:3,自引:0,他引:3
为建立一条简便、有效的构建哺乳动物细胞随机RNAi文库技术路线 ,首先通过PCR将 1 9~ 2 1ntsiRNA编码序列及终止信号等元件引入U6启动子下游 ,再将该扩增产物定向克隆至H1启动子的下游。构建的双启动子载体中 ,U6和H1相对排列 ,均以其间插入的靶序列为模板 ,分别转录出其正义和反义链 ,形成功能性siRNA。以EGFP和bcl-2为靶基因的实验表明 ,该方法构建的载体可有效地干扰相应基因的表达。 相似文献
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Ekaterini Iordanou Rachana R. Chandran Nicholas Blackstone Lan Jiang 《Journal of visualized experiments : JoVE》2011,(50)
Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.Download video file.(52M, mov) 相似文献
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目的:构建及筛选高效率针对早期生长反应基因-1(Egr-1)进行RNA干扰(RNAi)的质粒.方法:根据Egr-1基因mRNA序列,设计有小发夹结构的3条寡核苷酸序列,克隆到空栽体pGCSIL-GFP中,构建重组质粒,同时设计构建不针对任何特异基因的质粒作为阴性时照.将shRNA袁达质粒转粢HEK293细胞.通过对GFP表达量的观察,荧光定量PCR及western blotting定量检测Egr-1基因的表达,鉴定shRNA表达质粒对Egr-1的干扰效率.结果:针对小鼠Egr-1基因进行RNAi的3个序列中,有1个序列的干扰效率大于70%以上.结论:成功构建了1个针对小鼠Egr-1基因的高效RNAi表达质粒. 相似文献
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目的:建立在鸭胚成纤维细胞(DEF)中进行RNA干扰(RNAi)的技术平台,为鸭基因组功能的研究提供新的技术手段。方法:以绿色荧光蛋白(GFP)基因为报告基因,脂质体转染化学合成的GFP特异小干扰RNA(GFP-siRNA),用流式细胞仪测定GFP-siRNA对重组腺病毒(Adv-GFP)介导的GFP基因在DEF中表达的干扰效果。结果:200MOI(感染复数)Adv-GFP介导的GFP基因在DEF中表达效率最高,为31.20%±3.1l%,对DEF的活力无明显影响;GFP-siRNA能有效干扰GFP基因在DEF中的表达,相对抑制率为98.56%。结论:在DEF中进行RNAi是可行的,Adv-GFP是介导外源基因在DEF中表达较为理想的载体;首次建立了在DEF中进行RNAi的技术平台,为鸭基因组的功能等研究提供了新的技术手段。 相似文献
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用聚乙烯亚胺反向转染siRNA表达盒进行RNA干扰的研究 总被引:3,自引:0,他引:3
目的:建立适用于大规模RNA干扰(RNAi)筛选的siRNA生成与导入技术。方法:以绿色荧光蛋白(GFP)为模型,用PCR方法生成了包含U6启动子、互补的反义链和正义链以及终止序列的特异性siRNA表达盒(SEC),对聚乙烯亚胺(PEI)介导的转染SEC的方法及其效率进行研究。结果:PEI对细胞的毒性呈剂量依赖关系,当PEI与DNA的N/P=7.53,即PEI与DNA等量时,转染效率达到最高。PEI与脂质体LipofectAMINETM2000的转染效率无显著差异(P>0.05),对SEC的转染效率显著高于质粒载体(P<0.01)。反向转染的转染效率显著高于常规转染(P<0.01)。利用PEI将表达GFP特异性siRNA的SEC反向转染可稳定表达GFP的HeLa细胞株(HeLa-EGFP),荧光染色和Western印迹检测均表明可显著抑制GFP的表达。结论:PEI介导的SEC反向转染具有简便、快捷、经济的优点,可满足大规模RNAi筛选的需要。 相似文献