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。     

Construction of a cDNA library of Aphis gossypii Glover for use in RNAi

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site‐specific recombination of bacteriophage λ. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of 8.4 × 10⁶ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus‐induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi‐induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.     

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In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments.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.     

A construct with fluorescent indicators for conditional expression of miRNA

Background  

Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.     

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.     

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。     

The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

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。