PCBP1基因RNAi序列的鉴定及其干扰效应的初步研究

以能与PCBP1mRNA特异结合的核苷酸片段(21个碱基对)构建shRNA真核表达载体并稳定转染Hela细胞,通过半定量RT-PCR、免疫印迹和间接免疫荧光实验证实转染细胞中PCBP1的mRNA水平和蛋白质表达水平均被显著抑制.接着,本研究通过测定稳定转染Hela细胞的生长曲线、血清依赖性生长曲线和软琼脂集落形成率,证明PCBP1的沉默能够显著抑制细胞的生长增殖能力以及克隆形成潜能,揭示PCBP1可能直接或间接参与细胞周期的调控.本研究成功鉴定了沉默PCBP1基因表达的有效干扰片段,为下一步利用该有效干扰片段进行体内研究奠定了基础.同时,本研究的结果将有助于深入认识PCBP1蛋白在细胞周期调控和细胞癌变中的作用机制.     

沉默Vsx1基因后金鱼胚胎的差异蛋白质组学研究

Vsx1基因是与金鱼眼睛空间模式形成相关的调控基因和二倍体依赖型机制的候选目标基因.为进一步探究Vsx1在金鱼胚胎发育过程中的功能及其调控方式,采用反义寡核苷酸技术抑制Vsx1的表达,应用双向凝胶电泳(2-DE)进行蛋白分离,经PDQuest软件图谱分析,质谱分析初步鉴定,获得了Vsx1沉默引起的13个差异蛋白质.如Prohibidn-2(PHB2)、Nme2 protein等,并对这些差异蛋白质可能直接或间接地受到Vsx1的调控机理和Vsx1在金鱼胚胎发育过程中的功能进行了初步的理论分析,为进一步阐明Vsx1的功能和调控机制奠定了良好的基础.图3,表2,参17.     

磷硫代siRNA的抗肿瘤基因沉默活性研究

由于目前尚无高效合成立体特异性磷硫代siRNAs(PS-siRNAs)的化学体系,本研究针对潜在的癌症治疗靶点PLK1,用a-磷硫代三磷酸腺苷(ATPaS)、a-磷硫代三磷酸胞苷(CTPaS)和a-磷硫代三磷酸尿苷(UTPaS)通过T7RNA聚合酶转录合成了部分磷硫代修饰的Rp-磷硫代siRNAs(Rp-PS-siRNAs),探究了nat-siRNAs和PS-siRNAs血清稳定性和基因沉默活性的差异性。发现酶促合成的磷硫代siRNA几乎不影响siRNA的基因沉默效率,但却显著提高siRNA的血清稳定性。因此,酶促转录合成的Rp-PS-siRNA可望作为siRNA的修饰形式,以延长siRNA的生物活性半衰期,使siRNA广泛应用于生物医学临床研究领域。     

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.     

Mechanisms of gene silencing by double-stranded RNA

Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.     

Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.     

DNA序列拼接中重复序列屏蔽的一种新方法

针对序列拼接中的重复序列问题,提出了一种基于快速沃尔什变换的重复序列屏蔽方法．根据快速沃尔什变换的特点,快速给出重复序列所在的可能位置信息,从而快速识别重复序列且加以屏蔽．该方法不仅识别重复序列的错误率低而且大大降低了cPu运行时间,计算也简单易行,最后给出了模拟分析．     

基因沉默抑制子HC-Pro表达载体的构建

RNA沉默广泛存在于植物等大多数真核生物中,能够防御外源基因的入侵和调控基因表达等.然而,基因沉默抑制子HC-Pro蛋白的具体功能的研究并不十分清楚.本文重点介绍了基因沉默抑制子HC-Pro的表达载体pBI121-HC-Pro的构建及HC-Pro基因在烟草(Nicotiana benthamiana)中的稳定表达.并利用半定量RT-PCR技术检测了目的基因HC-Pro的表达,检测结果表明我们获得了HC-Pro基因稳定表达的转基因烟草株系,为进一步深入研究基因沉默抑制子HC-Pro的功能奠定了实验基础.     

磷硫代siRNA的YAP基因沉默活性研究

为了探究RNA聚合酶酶促合成非对映体纯PS-siRNA（pPS-siRNA）的基因沉默活性,本研究针对潜在的癌症治疗靶点Yes相关蛋白（YAP）,通过qPCR、Western blot等方法研究了其对YAP基因的沉默效果和对HeLa细胞的细胞毒性. 结果表明,与未修饰的siRNA（PO-siRNA）相比,pPS-siRNA可以更好地下调YAP基因表达（效率提高约30％）,而没有明显的细胞毒性. 此外,MTT细胞增殖实验与流式细胞术的结果表明,经pPS-siRNA敲降YAP后,HeLa细胞的增殖受到了抑制. 说明pPS-siRNA在基因治疗方面具有优越性以及YAP作为肿瘤治疗靶点的潜力.     

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．     

Plant polar growth in tobacco disturbed by y-tubulin gene silencing

To further understand the functions of y-tubulin in plant cells, we conducted a study in which the y-tubulin gene was down-regulated in tobacco plants (obtained by the Agrobacterium-mediated method). This involved transforming the target fragments, in which the sense and antisense partial y-tubulin cDNA fragments were ligated together, into Nicotiana tabacum var. Samsun NN. The y-tubulin down-regulated transformants developed multiple meristems or branches with trumpet-shaped leaves; their root generation also appeared abnormal, with the taproots undeveloped, whereas lateral roots were developed. In addition, the content of indole-3-acetic acid (IAA) and expression of polarity transportation vector PGPI were aberrant. These results suggest that y-tubulin gene silencing disturbed the polar growth of tobacco plants, and that this phenomenon was probably correlated with the IAA content and the polar transpor-tation process.     

Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast

Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally. Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4-10). Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast. Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes. Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding and the effects of loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.