A comparison of methods for successful triggering of gene silencing in Coprinus cinereus

Abstract

Post-transcriptional gene-silencing methods (PTGS), including RNAi, are becoming increasingly pervasive in functional genomics. To advance analysis of the recently sequenced Coprinus cinereus genome, a high throughput gene silencing method is essential. We have exploited the GFP reporter gene to evaluate and quantify efficacy of three different silencing strategies. Modular constructs that encompassed antisense, untranslatable sense, and RNAi-mediating hairpin sequences, were transformed into a GFP-expressing host strain. Transformants exhibiting strong downregulation and partial suppression of GFP were recovered with all three constructs. Analyses of protein and transcriptional nucleic acids revealed that the antisense and hairpin sequences yielded similar levels of GFP suppression, and were both more efficient than untranslatable sense sequences. Our antisense vectors will expedite functional characterisation of C. cinereus and the modular nature of the constructs should permit exploitation of directional cDNA libraries for high throughput screening.

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

“…PG78Green was previously described (Heneghan et al, 2007), expresses GFP and was used as host strain for pSUPER transformations. C. cinerea strains were maintained at 37ºC on complete medium (CM) (Lewis, 1961) supplemented with 100 μg/L L-tryptophan or 2 μg/L para-aminobenzoic acid as required.…”

“…GFP activity was fluorimetrically quantified as previously described (Heneghan et al, 2007) using three independent protein extractions for each strain.…”

“…Coprinopsis cinerea is a model homobasidiomycete used in many studies such as sexual development and meiosis, and also to exploit tools for functional genomics in filamentous fungi (Heneghan et al, 2007;Namekawa et al, 2005;Wälti et al, 2006).…”

“…The study of gene function in filamentous fungi has been hampered by the lack of efficient tools but RNA mediated gene silencing has recently been successfully achieved in these fungi (Heneghan et al, 2007;Namekawa et al, 2005;Wälti et al, 2006;De Jong et al, 2006). Most of these studies have involved laborious and time consuming multiple-steps to clone target sequence in sense and antisense orientations to yield dsRNA forming hairpin constructs that initiate RNAi mediated gene silencing.…”

Wood production, wood technology, and biotechnological impacts

“…They are however not essential as recently shown by gene inactivation in a self-compatible homokaryon through the posttranscriptional acting RNAi technique that leads to degradation of gene transcripts (Wälti et al 2006). Establishing this technique in C. cinerea in various laboratories (Namekawa et al , Heneghan et al 2006, Wälti et al 2006) is a great achievement for analysis of those genes for which mutants are not available. The gene for the LIM15/DCM1 recombinase has been inactivated in a self-compatible homokaryon with the result of an abnormal chromosomal pairing in meiosis, a highly reduced basidiospore number and viability (Namekawa et al 2005).…”