title: 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.
authors.affiliation: School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK.
authors.forename: Mary N
authors.initials: MN
authors.lastname: Heneghan
authors.forename: Ana M S B
authors.initials: AM
authors.lastname: Costa
authors.forename: Michael P
authors.initials: MP
authors.lastname: Challen
authors.forename: Peter R
authors.initials: PR
authors.lastname: Mills
authors.forename: Andy
authors.initials: A
authors.lastname: Bailey
authors.forename: Gary D
authors.initials: GD
authors.lastname: Foster
mesh_terms: D001483:Base Sequence; D003302:Coprinus / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins