转录后水平的基因沉默--RNA干涉

RNA干涉（RNAinterference,RNAi)是1998年首次在秀丽线虫中发现并证明属于转录后水平的基因沉默（PTGS）机制，它利用双链RNA（dsRNA)特异性地降解相应序列的mRNA成为siRNA，从而特异性地阻断相应基因的表达，广泛存在于从真菌到植物。从无脊椎动物到哺乳动物的各种生物中，本文介绍了基因沉默的机理，RNA干涉的分子机制及技术应用等方面的进展。RNA干涉在后基因组时代的基因功能研究和药物开发中将具有广阔的发展前景。     

RNA干涉和基因沉寂

RNA干涉是新近认识到的一种自然现象，但已被作为一种研究基因功能的有效工具，广泛运用于植物、真菌、线虫、果蝇以及哺乳动物。随着人们对RNA干涉机制认识的不断深入，RNA干涉技术将不断完善并应用到更为广阔的领域，为人类揭示生物界功能基因组功能的奥秘作出突出贡献，同时为人们探索基因治疗提供新的思路。     

RNA干涉和基因沉寂

RNA干涉是新近认识到的一种自然现象 ,但已被作为一种研究基因功能的有效工具 ,广泛运用于植物、真菌、线虫、果蝇以及哺乳动物。随着人们对 RNA干涉机制认识的不断深入 ,RNA干涉技术将不断完善并应用到更为广阔的领域 ,为人类揭示生物界功能基因组功能的奥秘作出突出贡献 ,同时为人们探索基因治疗提供新的思路     

寄生线虫功能基因组学的研究进展

寄生线虫病严重危害人和动物的健康,但目前防治寄生线虫病尚存在困难。随着分子生物学技术的迅速发展,运用生物技术手段来控制寄生线虫病将成为可能,由此而来,研究寄生线虫的基因功能具有非常重要的意义。本文简要概述了研究寄生虫功能基因组学的一些方法,如研究差异表达基因的方法、RNA干涉技术、寄生虫的转基因技术和生物信息学等,并综述了猪蛔虫、圆线科线虫、旋盘尾丝虫、旋毛虫、马来布鲁线虫和有齿食道口线虫等一些重要寄生线虫在功能基因组学上的研究进展。     

RNA干涉：沉默基因的机制及应用研究新进展

RNA干涉 (RNA interference,RNAi)是一种由双链 RNA(double- stranded RNA ,ds RNA)始动的序列特异性基因沉默机制 ,广泛存在于各种生物 ,乃至人类。对 RNAi分子机制及生物学意义研究不断深入 ,已发现了许多参与 RNAi的基因和蛋白质 (酶类 ) ,并证明了 RNAi与细胞分化及生物发育密切相关。同时 RNAi也是一种研究基因表达调控和基因功能强有力的工具。本文就 RNAi的分子机制、生物学意义及其应用研究方面的新进展做一介绍     

RNA干涉：沉默基因的机制及应用研究新进展

RNA干涉（RNAinterference,RNAi)是一种由双链RNA（double-strandedRNA,dsRNA）始动的序列特异性基因沉默机制，广泛存在于各种生物，乃至人类；对RNAi分子机制及生物学意义研究不断深入，已发现了许多参与RNAi的基因和蛋白质（酶类），并证明了RNAi与细胞分化及生物发育密切相关。同时RNAi也是一种研究基因表达调控和基因功能强有力的工具，本就RNAi的分子机制，生物学意义及其应用研究方面的新进展做一介绍。     

人与小鼠染色体的同源性及遗传病动物模型

近二十余年来,人类及其他哺乳动物,特别是小鼠基因图的研究取得了很大进展。哺乳类基因功能的研究表明,即使是亲缘关系很近的种间,基因顺序和染色体结构也有很大差异;但另一方面,种间存在着进化过程中保留的连锁群。由于人类生育繁殖的特点,在某种程度上限制了人类遗传学的发展,对人类以外的灵长类的研究已在比较遗传学及比较基因制图方面得到了不少资料。但是,研究条件上存在一定困难。目前对小鼠染色体核型和基因已有较多了解,并且在研究上有许多便利之处。因此,比较人类与小鼠染色体结构、基因及基因功能的相互关系或异同点,建立人类重要遗传病的小鼠模     

RNA干涉技术在功能基因组学和医学研究中的应用

RNA干涉 (RNAi)能特异性降解 m RNA,在转录后水平抑制基因活动 ,作为新的技术平台将对功能基因组学研究和疾病的防治产生重要影响。RNAi能特异地“剔出”靶基因的表达及功能 ,可广泛地运用于基因功能的分析。 RNAi是动植物在抗御病毒、转座子等外来基因侵袭或内源性基因变异的进化过程中发展起来的 ,利用这种内在的防御机制可望发展成为征服感染、肿瘤等疾病的有效的手段。     

反义RNA技术的应用与进展

反义 RNA技术作为反义核酸技术之一 ,已在越来越广泛的领域得到应用。本文在与反义寡核苷酸技术进行比较的基础上 ,系统地介绍了反义 RNA技术方法及在各领域的应用。相信随着对基因功能研究的广泛开展及基因治疗研究的深入 ,反义 RNA技术必将成为一种有力的工具 ,在基因功能研究方面 ,特别是在后基因组研究中做出巨大贡献     

RNA干涉文库的设计与应用

人类基因组计划的巨大发展提供了大量原始生物学信息,但对其功能信息的了解还相当有限。RNA干涉技术为研究基因功能提供了一种快速而经济的方法。构建靶向大量基因的RNA干涉文库在基因组范围内实现基因的沉默,已成为研究细胞生物学行为的一种新的功能基因组学研究方法。近年来相继出现许多针对RNA干涉文库的设计策略,并在一定规模内成功实现遗传学筛选,为将RNA干涉文库用于功能基因组学的研究进行了意义深远地探索与尝试。     

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.     

Interaction of viruses with the mammalian RNA interference pathway

It has been known for some time that plants and insects use RNA interference (RNAi) as nucleic acid-based immunity against viral infections. However, it was unknown whether mammalian cells employ the RNA interference pathway as an antiviral mechanism as well. Over the past years, it has become clear that a variety of viruses, first in plants but recently in insect and mammalian viruses as well, encode suppressors of the RNAi pathway arguing for an antiviral role of this machinery. More recent findings have revealed that certain viruses encode their own microRNAs or microRNA-like RNA molecules, which are processed by the mammalian RNAi machinery. Furthermore, host-encoded microRNAs have been shown to both silence and enhance intracellular levels of viral RNAs. These findings argue that interactions between the RNAi pathway and viral genomes can profoundly affect the outcomes of the viral life cycles and contribute to the pathogenic signatures of the infectious agents.     

RNA干扰在哺乳动物细胞高通量基因功能研究中的应用

RNA干扰(RNAi)技术是近年来迅速发展起来的一种高效、特异、易操作的基因阻断技术,它已被应用于线虫、果蝇等低等生物的大规模功能丧失表型遗传学筛选中,并成功鉴定了大量的新基因。近期,在利用现有siRNA文库进行的小规模哺乳动物细胞功能基因筛选研究中,RNAi同样显示了卓越的性能。随着高复杂度的siRNA文库的构建和筛选策略的完善,RNAi在哺乳动物细胞高通量基因功能研究中的应用已日见成熟。     

L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism

Long Interspersed Element 1 (L1) is a retrotransposon that comprises ∼17% of the human genome. Despite its abundance in mammalian genomes, relatively little is understood about L1 retrotransposition in vivo. To study the timing and tissue specificity of retrotransposition, we created transgenic mouse and rat models containing human or mouse L1 elements controlled by their endogenous promoters. Here, we demonstrate abundant L1 RNA in both germ cells and embryos. However, the integration events usually occur in embryogenesis rather than in germ cells and are not heritable. We further demonstrate L1 RNA in preimplantation embryos lacking the L1 transgene and L1 somatic retrotransposition events in blastocysts and adults lacking the transgene. Together, these data indicate that L1 RNA transcribed in male or female germ cells can be carried over through fertilization and integrate during embryogenesis, an interesting example of heritability of RNA independent of its encoding DNA. Thus, L1 creates somatic mosaicism during mammalian development, suggesting a role for L1 in carcinogenesis and other disease.     

siRNA抑制外源绿色荧光蛋白在neuro-2a细胞株中的表达

目的：用RNAi技术抑制哺乳动物神经元中外源报告基因的表达，并探讨该过程中RNAi的时间及剂量效应，为利用RNAi技术研究神经元基因功能提供参考。方法：采用神经细胞来源的细胞系neuro-2a小鼠神经瘤细胞为模型，将外源绿色荧光蛋白（GFP）基因转染到neuro-2a细胞内，利用体外转录法合成siRNA，分为3个剂量组对其进行干扰。结果：Neuro-2a细胞可以被有效地转染，而且siRNA在neuro-2a细胞内可以有效发挥干扰作用。这种干扰作用呈现出一定的剂量效应。结论：RNAi技术可以成功用于neuro-2a细胞抑制基因的表达，这种对GFP表达规律及RNAi剂量效应的研究为利用RNAi技术研究神经元基因功能提供了一定的理论参考和依据。     

Attenuation of SARS coronavirus by a short hairpin RNA expression plasmid targeting RNA-dependent RNA polymerase

Severe acute respiratory syndrome (SARS) is a highly contagious and sometimes a lethal disease, which spread over five continents in 2002-2003. Laboratory analysis showed that the etiologic agent for SARS is a new type of coronavirus. Currently, there is no specific treatment for this disease. RNA interference (RNAi) is a recently discovered antiviral mechanism in plant and animal cells that induces a specific degradation of double-stranded RNA. Here, we provide evidences that RNAi targeting at coronavirus RNA-dependent RNA polymerase (RDRP) using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Moreover, this construct significantly reduced the plaque formation of SARS coronaviruses in Vero-E6 cells. The data may suggest a new approach for treatment of SARS patients.     

Silencing of African horse sickness virus VP7 protein expression in cultured cells by RNA interference

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA. Short interfering RNAs (siRNAs) are the mediators of RNAi and represent powerful tools to silence gene expression in mammalian cells including genes of viral origin. In this study, we applied siRNAs targeting the VP7 gene of African horse sickness virus (AHSV) that encodes a structural protein required for stable capsid assembly. Using a VP7 expression reporter plasmid and an in vitro model of infection, we show that synthetic siRNA molecules corresponding to the AHSV VP7 gene silenced effectively VP7 protein and mRNA expression, and decreased production of infectious virus particles as evidenced by a reduction in the progeny virion titres when compared to control cells. This work establishes RNAi as a genetic tool for the study of AHSV and offers new possibilities for the analysis of viral genes important for AHSV physiology.