RNAi技术及其应用

RNAi技术是指向特异的靶基因中导入双链RNA从而导致靶基因沉默的一种新技术.RNAi在调节转录后的基因表达水平方面有其独特的作用特点,随着研究的深入,具有广阔的应用前景.用RNAi技术调节基因表达在对不同生物基因功能的研究中已广泛应用.文中简要综述了RNAi技术的研究历史、研究进展和应用前景.     

RNA干扰及其应用

RNA干扰（RNA interference，RNAi）是真核生物中普遍存在的一种自然现象，是由双链RNA（double—stranded RNA，dsRNA）启动的序列特异的转录后基因沉默过程．在这一过程中，双链RNA被核酸内切酶切割成小干扰RNA（small interfering RNA，siRNA），小干扰RNA与相关的蛋白质结合成RNA诱导沉默复合体（siRNA—induced silencing complex，RISC），RISC识别并降解靶mRNA．现在认为RNA干扰是真核生物共有的抗病毒、抗转座子、抗转基因和抗异常RNA的基因组免疫系统．随着RNA干扰的研究，产生了一种新的技术-RNA干扰技术，并得到了广泛的应用．     

RNAi在植物中的研究进展

RNAi（RNA interfering）是由靶基因同源的双链RNA引发的在动植物中普遍存在的序列特异性转录后基因沉默。最早在植物体中发现,现已成为后基因组时代的重要研究手段。对RNAi在植物功能基因组、生长发育、抗病毒、品质改良方面的最新研究进行了综述,展望了今后的发展前景。     

RNAi技术及其在基因组学研究中的应用

RNA干扰（RNA interference，RNAi）是一种真核生物体内的基因调控机制．在介绍RNAi的作用机制及作用特点的基础上，综述了其在基因功能研究、基因组免疫系统、人类疾病的基因治疗及水产养殖病害的防治等基因组学研究领域的最新进展与应用．     

RNA干涉与基因沉默研究进展

生物体内导入双链RNA(dsRNA)后会引起体内同源基因特异的沉默，这种现象称为RNA干涉(RNAi)．基因沉默是生物体基因表达调控的一种重要机制，具有重要生物学功能．就RNA干涉与基因沉默的研究历史、作用机制和应用作了综述．     

RNAi及其在生命科学研究中的应用

RNAi即RNA干扰(RNA interfering),是近几年才发现的一种由双链RNA引起的基因沉默的现象,是目前分子生物学领域研究的热点。介绍RNAi的发现,并对RNAi在生命科学研究领域的最新应用进行综述。     

RNA干涉研究进展

RNA干涉(RNAi)是由双链RNA(dsRNA)引起的序列特异性基因沉默,这是目前研究的热点,具有广阔的应用前景.对RNAi的分子机制、生物功能、研究策略及RNAi技术在基因功能、基因治疗、植物品质改良等方面的应用进行了综述.     

RNAi技术的研究进展及其应用

RNA干扰（RNAi）是指双链RNA引起的mRNA水平互补序列基因表达的关闭,即序列特异性转录后基因沉默,是生物体进化过程中抵御外未基因和病毒感染的进化保守机制。本文对RNA干涉现象的发现,作用机制及各方面应用作了简单的论述。     

番茄红素β-环化酶基因（LcyB）启动子调控LcyB RNAi双元载体构建

根据番茄基因组DNA序列信息设计引物进行PCR扩增了Micro-Tom中番茄红素-环化酶（Lycopene -cyclase, LcyB）基因起始密码子上游1 534 bp启动子区域序列（LcyBp）,生物信息学分析表明,该启动子序列中存在TATA-盒、CAAT-盒、昼夜节律响应元件Circadian、光响应元件Box I、真菌激发子响应元件Box-W1、低温响应元件LTR、响应赤霉素的作用元件P-box、乙烯响应元件ERE、响应生长素的作用元件TGA-element等顺式作用元件. 依据番茄LcyB基因序列,设计2对含有不同酶切位点的特异引物进行PCR扩增LcyB基因3端特异的276 bp DNA片段,利用RNAi载体pKANNIBAL构建了LcyB启动子-LcyB基因正义片段（Sense）-PDK内含子-LcyB基因反义片段（Antisense）-OCS终止子的RNAi表达框,并将这一RNAi表达框插入植物双元表达载体pART27的Not I位点,构建成本研究的LcyB启动子驱动的LcyB基因RNAi植物双元表达载体pART-LcyBp-RNAi-LcyB. 为利用RNAi技术特异性敲除LcyB基因进而提高番茄果实中番茄红素含量奠定实验基础.     

RNAi技术研究进展

RNAi是由双链RNA引起的基因沉默现象，它通过降解具有同源序列的mRNA起作用，特殊设计的siRNA能使目的基因发生特异性沉默．目前，获得siRNA已经非常方便，导入方式最常用的是微注射．RNAi技术有编码区RNAi和启动子区RNAi两大类，均可以产生类似基因敲除的功能。在家蚕功能基因的研究中已经使用了这种方法．随着RNAi技术的不断进步，RNAi可广泛地应用到功能基因组学，药物靶点筛选，疾病治疗等方面．     

RNAi-mediated gene silencing in non-human primates

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.     

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.     

Identification of RPS14 as a 5q- syndrome gene by RNA interference screen

Somatic chromosomal deletions in cancer are thought to indicate the location of tumour suppressor genes, by which a complete loss of gene function occurs through biallelic deletion, point mutation or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis. In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Here we describe an RNA-mediated interference (RNAi)-based approach to discovery of the 5q- disease gene. We found that partial loss of function of the ribosomal subunit protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells, and also that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-ribosomal RNA in RPS14-deficient cells that is functionally equivalent to the defect in Diamond-Blackfan anaemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital syndrome causing bone marrow failure. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function and suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.     

RNA干涉及其在肿瘤研究中的应用

RNAi是双链RNA介导的转录后基因沉默的过程，是一种高效的高特异性抑制基因表达的新途径。通过双链小干涉RNA(siRNA)与一系列蛋白质结合形成siRNA诱导的沉默复合体(RISC)并活化，然后，RISC对靶基因进行识别、降解。与反义方法相比，siRNA具有更好的抑制效果。RNAi的应用将为癌症的基因治疗提供新的方法。     

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.     

A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys?9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.