Mechanisms and Application of Gene Silencing in Oomycetes
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This chapter contains sections titled: Introduction Gene Silencing—The Basic Parts List Mechanisms of Silencing Gene Silencing in Oomycetes: Tales from the Laboratory and Clues from Genomes Strategies for Application of Gene Silencing in Oomycetes Validation: Linking Gene Silencing to Phenotype Which Strategy? Stable versus Transient Gene Silencing Conclusions and Scope for Future Work Acknowledgments ReferencesKeywords:
RNA-induced silencing complex
SummaryRNA interference (RNAi) has become a powerful functional genomics tool that can be used to effectively silence gene expression. The implications for analysis of loss-of-function phenotypes through systemic or localized silencing are enormously significant in the application of this technology. The Sid-I gene was implicated in the cellular import of RNAi signal that enables passive uptake of dsRNA. Here we demonstrate that RNAi in the honey bee (Apis mellifera) is systemic and our data suggest that honey bee SID-I homologue, a putative transmembrane protein encoded by AmSid-I, is necessary for the uptake of systemically administered dsRNA and subsequent gene silencing.The honey bee SID-I homologue shares strong similarities with human (NP-060169; 44.3%), mouse (NM-198034; 43.9%), and Caenorhabditis elegans (Q9GZC8; 19%).AmSid-I was expressed in the entire set of honey bee tissues examined with the highest abundance in adult head followed by egg tissue. To test the role of AmSid-I in the systemic effect of RNAi, we induced systemic gene silencing of the honey bee Toll-related receptor 18W by a feeding-soaking delivery method of dsRNA and measured expression levels of AmSid-I and Am18w using real time PCR. A 3.4–fold increase in expression of AmSid-I was observed at 26 h. In contrast, Am18w gene expression was decreased about 60–fold at 30 h. High mortality and morphological abnormalities were also seen due to gene silencing. The presence of SID-I in honey bees and its function as a transmembrane channel that facilitates uptake of dsRNA are discussed.
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RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy.
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In this thesis we studied various aspects of siRNA mediated silencing. siRNA mediated silencing is initiated by the introduction of dsRNA, transgenes and viral infection. Our first goal was to study the ability of the core pathway of RNA silencing to explain transgene and dsRNA induced silencing. To that extend we developed and studied concise models of the RNA silencing pathway. Secondly, we investigated the efficacy of RNA silencing to reduce viral infections, and added a replicating RNA virus. While we initially studied antiviral silencing within the cell, we extended our study to tissue level dynamics and symptom development in plants. Finally, we added virus encoded silencing suppressors and studied the effect and capability of the different silencing suppressors. We found that the available knowledge about the RNA silencing pathway was not sufficient to explain transgene induced silencing. We proposed mechanisms that could result in transgene induced silencing and recently the existence of these mechanisms has been confirmed experimentally. Our detailed model of both intra- and inter-cellular dynamics gives an explanation for the observed patterns on plant leaves that occur during viral infection. These patterns can have the form of local spots, concentric circles, or resemble mosaic-like patterns. We have shown that models studies are indispensable to understand complex cellular pathways. On the one hand we have shown that the generally accepted pathway of RNA silencing was insufficient to explain the experimental findings on transgene induced silencing it was supposed to explain. On the other hand we have shown that the interaction of RNA silencing and viral growth is indeed able to generate infection patterns for which the link to RNA silencing was still tentative.
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RNA-induced silencing complex
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RNA-induced silencing complex
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RNA-induced transcriptional silencing
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RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.
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RNA 干扰(RNAi ) ，为 post-transcriptionalgene silencing 的最新发现的方法之一，广泛地被用来为介绍一个 RNA silencing 信号进植物通过转基因的方法调查基因函数。在现在的学习，我们构造了， adexamethazone (DEX ) 怀有特定的顺序碎片(168-bp ) 的可诱导的 RNAi 二进制向量对 KatB 和 KatC 相应， Arabidopsis 的二 kinesin isoform 基因，它被证明在导致DEX 的转基因的工厂导致 KatB 和 KatC 的 post-transcriptional 基因 silencing 。转基因的同型结合的 Arabidopsis 上的 RT-PCRand 北污点分析(作为 RNAi 类型工厂称为) 证明那 DEX 动机引起 KatB 和 KatC mRNA 降级。与一个简化方法， Arabidopsisgrafting 有效地在 RNAi 类型和野类型的线之间被执行。目标基因 mRNAlevels 基于半量的 RT-PCR 被测试。我们的结果证明 DEX-inducedgene silencing 信号能在野类型的根茎或接穗在 KatB 和 KatC mRNA 导致减小，显示 RNAi 的 silencing 信号能越过接枝连接双向地被播送 RNAi 种是否是接穗或股票。与 grafted 烟草上的以前报导的结果相对照， post-transcriptional 基因 silencing 信号的传播在 grafted Arabidopsis 由 RNAi 引起了在烟草是比那更有效的。
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RNA interference (RNAi) is a posttranscriptional gene-silencing event in which short double-stranded RNA (siRNA) degrades target mRNA. Because of its potent and highly specific gene-silencing effect, RNAi is expected to be used in the treatment of various diseases. Cancer is one of the major targets of RNAi-based therapy, because silencing oncogenes or other genes contributing to tumor progression can be target genes for RNAi. The delivery of RNAi effector to target cells is one of the key factors determining therapeutic efficacy, because gene silencing is limited to cells reached by RNAi effectors. Tumor cell lines stably expressing reporter genes were confirmed to be effective in sensitively and quantitatively evaluating RNAi effects in tumor cells in vitro and in vivo. Quantitative analyses of the gene-silencing effect revealed that short-hairpin RNA expressing plasmid DNA (pshRNA) has more durable effects than siRNA. Intratumoral injection of RNAi effectors was effective in suppressing target gene expression in tumor cells, and silencing of β-catenin or hypoxia-inducible factor-1α (HIF-1α) significantly inhibited tumor growth. RNAi effectors were successfully delivered to tumor cells colonizing the liver through the vascular route. We found that tumor-bearing liver showed elevated HIF-1α expression in the cells, and the silencing of the expression in normal liver cells is also effective in inhibiting metastatic tumor growth. These results indicate the possibility of RNAi-based cancer therapy.
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The α-synuclein (SNCA) gene is a responsible gene for Parkinson's disease (PD); and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi); however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs) that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named "expression-control RNAi" (ExCont-RNAi). ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.
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Gene silencing by RNA interference (RNAi) allows, for the first time, investigation of the balance between the life and death of a cell under stress-free conditions. Thus it is now possible to (i) identify key antiapoptotic genes that constitutively enable, for example, cancer cell survival, and (ii) map the pathways such genes control (using RNAi co-silencing). New gene targets for anticancer therapy are identified and, full circle, RNAi provides the means for their selective therapeutic silencing.
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