RNAi-Based Gene Silencing Using a GFP Sentinel System in Histoplasma capsulatum
RNA interference (RNAi) has revolutionized reverse genetics in eukaryotic organisms, particularly those in which homologous recombination is inefficient or impractical. The ability to deplete or knock-down a targeted gene product without requiring genetic
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1. Introduction The ability to manipulate gene function is fundamental in understanding how a gene contributes to the phenotype of an organism. Reverse genetic analysis is a direct approach that can be used to establish the role of each gene; but in Histoplasma capsulatum, as in most pathogenic fungi, generating gene knock-outs by homologous recombination is difficult, time consuming, and often unsuccessful. RNA interference (RNAi) is an effective alternative that can be used to deplete a gene product without requiring molecular disruption of the targeted gene. The methodology for triggering RNAi overcomes many of the technical obstacles associated with creating gene deletions and offers a quicker and more efficient means of assessing the function or role of the targeted gene. Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_10, © Springer Science+Business Media, LLC 2012
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B.H. Youseff and C.A. Rappleye
The normal cellular function of RNAi is believed to be an evolutionarily conserved defense mechanism of the eukaryotic genome, protecting the genetic code from transposable elements and viruses (1). More recent studies have found RNAi also contributes to the regulation of native genes (2). RNAi is triggered by double-stranded RNA molecules which are processed by the cell’s RNAi machinery into short-RNA fragments that guide the RNAinduced silencing endonuclease complex (RISC) to the endogenous gene transcript in a sequence-specific manner (3). Destruction of the homologous mRNA by RISC prevents further synthesis of the gene product resulting in depletion or “knock-down” of the targeted gene function. The overall reduction in gene product levels results from the net contribution of new mRNA synthesis and degradation of transcripts by RNAi. Although depletion is never complete with RNAi, exploitation of this posttranscriptional interference mechanism to target a gene of interest often produces a phenotype similar to that resulting from gene deletion. In H. capsulatum, double-stranded RNAs can effectively trigger RNAi-based depletion of gene products (4). The double-stranded RNA trigger is generated in vivo by the transcription of inverted copies of a region of the targeted gene (see Fig. 1) to produce a stem-loop RNA molecule. In Histoplasma, double-stranded RNA stem lengths greater than 500 bp produce a significant degree of silencing (4). To generate this RNAi trigger in vivo, the construct is inserted into an RNAi vector which is linearized by restriction digest and transformed into Histoplasma yeast by electroporation. RNAi plasmids are transformed into ura5-mutant strains of Histoplasma and selection for plasmid-transformed yeast is achieved by virtue of the plasmid-encoded URA5 gene, which restores uracil prototrophy. The RNAi construct is placed downstream of the constitutive Histoplasma histone-2B promoter to produce high levels of the stem-loop RNA and thus the strongest RNAi effe
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