RNAi导致基因沉默的机制和应用进展

RNAi主要通过双链RNA(dsRNA)被核酸酶切割成21~23 nt的干涉性小的RNA,即siRNA,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现的；RNAi需要多种蛋白质因子以及ATP参与,而且具有生物催化反应特征；RNAi具有高效性和高度特异性,作为一种关闭特定基因的新技术,为基因功能研究提供了一个快速、简便的方法,在后基因组时代应用前景广阔。     

RNA干涉及其在蚕功能基因组研究中的应用

RNA干涉 (RNAInterferance ,简称RNAi)通过导入一段与内源靶基因同源的双链RNA(dsRNA)序列 ,使内源mRNA降解 ,从而达到阻抑基因表达的目的。已在线虫等生物中建立RNAi技术 ,对难于获得突变体的基因或生物体尤其有效。日本九州大学、东京大学和农业生物资源研究所的 3个研究小组对RNAi在家蚕中的应用进行了探索 ,初步发现在家蚕和其培养细胞中存在RNA干涉现象。探讨了RNA干涉技术在家蚕基因功能研究中应用的可能性。     

转基因小鼠中应用RNAi的初步探讨

RNA干扰(RNA interference,RNAi)是通过双链RNA介导,特异性地降解相应序列的mRNA,从而导致转录后水平的基因沉默。近年来,利用小分子RNA(small interfering RNA,siRNA)介导的RNAi技术已越来越多的应用于哺乳动物细胞中。同时,在活体中利用RNAi研究多种生物基因功能也成为一种有效的手段。文章旨在介绍在转基因小鼠中RNAi的应用。     

家蚕新的卵色相关基因Bmwh2的克隆及组织表达与功能研究

腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporter,ABC转运蛋白)是古老而庞大的家族,广泛分布于从细菌到人类各种生物体中,主要利用ATP水解释放的能量实现多种底物的跨膜主动转运。通过RT-PCR扩增、克隆测序了家蚕ABC转运子Bmwh2完整的开放阅读框。用生物信息学方法分析发现,Bmwh2有14个外显子和13个内含子,编码689个氨基酸残基,分子质量77.38 kD,等电点8.42;Bmwh2含有1个核苷酸结合结构域和6个α螺旋构成的跨膜结构域,为半转运子,属于家蚕ABC转运子超家族G亚族成员。利用半定量RT-PCR方法分析Bmwh2在家蚕5龄第3天幼虫不同组织的表达水平,以精巢中的相对表达量最高。用合成的siRNA显微注射胚胎发育时期的蚕卵,对Bmwh2进行干涉研究,获得了白卵和嵌合体的干涉表型。根据实验结果,推测Bmwh2可能参与家蚕浆液膜色素前体的转运。     

RNAi基因沉默的动力学研究进展

RNA干扰(RNAi)是一种由小双链RNA介导的转录后基因沉默现象。在RNAi发挥基因沉默效能的过程中,许多因素都会直接或间接的影响最终的沉默效率和沉默时间。论文从siRNA类型的选择、siRNA的递送和细胞内的RNAi途径3个方面对RNAi途径中各个影响其效能发挥的因素及其解决方法做一综述,同时对描述RNAi基因沉默动力学的数学模型及其应用进行了介绍。     

RNA干扰作用的研究进展

作为一种古老的生物体自我监督机制，RNA干扰（RNAi）通过双链RNA（dsRNA）作用于同源基因的特异性序列，使其沉默。而小干扰RNA（siRNA）则类似RNAi作用中的媒介物，能够沉默体细胞中的目的基因而不会激活dsRNA依赖性蛋白酶介导的非特异性抑制作用。目前，RNAi技术已被广泛应用于新基因的筛选、基因功能鉴定、信号转导研究以及基因治疗等方面。显示出了十分广阔的应用前景。     

RNA干涉机制研究进展

RNA干涉(RNAi)是近年来的研究热点,相关综述有很多。但随着研究的不断深入,RNAi的作用机制和影响变得越来越复杂,以致难以简单而准确地定义RNAi。新的研究发现主要有:(1)RNAi在异染色质形成中有重要作用,并影响染色质修饰的分布,如组蛋白H3K9甲基化和DNA甲基化。(2)在RNAi中会产生二级siRNA,使干涉效果增强;二级siRNA为不同的Argonaute家族蛋白所结合,其作用机制更加复杂。(3)新鉴定出具有RNAi作用的小RNA,如rasiRNA和piRNA,其产生和作用机制都与dsRNA和siRNA有所不同。此外,论文还介绍了RNAi在治疗癌症和人畜传染病等方面的应用,并对应用中出现的问题进行了讨论。     

RNA干扰及其在植物中的研究进展

RNA干扰(RNA interference,RNAi)是一种由双链RNA(double-stranded RNA,dsRNA)介导、能够特异沉默靶基因的转录后基因沉默现象,为植物基因功能的研究开辟了新途径.本研究主要综述了RNA干扰现象的发现、RNA干扰的过程及其特点、RNA干扰在植物中的诱导方法及载体构建、近年来R...     

RNA干扰的研究进展

RNA干扰(RNAi)在抵抗病毒感染、抑制转座子活动、调控内源性基因表达等方面发挥重要作用,其高特异性、高效性等显著优势将成为研究基因功能的全新手段.本文简要概括RNAi作用机制和siRNA技术的原理,同时也讨论了RNAi技术在各领域的应用.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.     

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.     

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.     

Research Progress on Small Interfering RNA and Its Delivery Systems

Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However,siRNA shows poor pharmacological properties, such as low serum stability,off-targeting, and innate immune responses, which present a significant challenge for clinical applications.In addition, siRNA cannot cross the cell membrane for siRNA activity because of its anionic property and stiff structure.Therefore, the development of a safe,stable,and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo, and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA,in order to lay a good foundation for the later research.