RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。     

Stable and heritable gene silencing in the malaria vector Anopheles stephensi

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.     

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.     

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.     

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites.     

Dynamics of gene silencing by RNA interference

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.     

Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.     

RNA干扰与抗病毒感染

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默(PTGS)现象,是一种古老的生物抵抗外在感染的防御机制。RNAi因其在维持基因组稳定、调控基因表达和保护基因组免受外源核酸侵入等方面发挥的重要作用,已被广泛用于探索基因功能、基因治疗和新药的研发。外源导入siRNA引发的RNAi可以特异性抑制病毒的复制与感染,为抗病毒感染治疗开辟了一条新的途径。     

Small RNAs from cereal powdery mildew pathogens may target host plant genes

Small RNAs (sRNAs) play a key role in eukaryotic gene regulation, for example by gene silencing via RNA interference (RNAi). The biogenesis of sRNAs depends on proteins that are generally conserved in all eukaryotic lineages, yet some species that lack part or all the components of the mechanism exist. Here we explored the presence of the RNAi machinery and its expression as well as the occurrence of sRNA candidates and their putative endogenous as well as host targets in phytopathogenic powdery mildew fungi. We focused on the species Blumeria graminis, which occurs in various specialized forms (formae speciales) that each have a strictly limited host range. B. graminis f. sp. hordei and B. graminis f. sp. tritici, colonizing barley and wheat, respectively, have genomes that are characterized by extensive gene loss. Nonetheless, we find that the RNAi machinery appears to be largely complete and expressed during infection. sRNA sequencing data enabled the identification of putative sRNAs in both pathogens. While a considerable part of the sRNA candidates have predicted target sites in endogenous genes and transposable elements, a small proportion appears to have targets in planta, suggesting potential cross-kingdom RNA transfer between powdery mildew fungi and their respective plant hosts.     

RNA干扰

RNA干扰（RNA interference,RNAi）现象是指,当与内源性mRNA编码区某段序列同源的双链RNA(dsRNA)导入细胞后,该mRNA发生特异性的降解,而导致该基因表达的沉寂。这可能反映了生物防范病毒或转座子诱导DNA突变的一种防御机制。RNA干扰已经成为一种重要的研究基因功能的有力工具,并且有希望在对疾病的防御及治疗中发挥重要的作用。