Costa, Ana M.S.B., Mills, P. R. (Peter R.), Bailey, Andy M., Foster, Gary D. and Challen, Michael P.. (2008) Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes. Journal of Microbiological Methods, Vol.75 (No.2). pp. 205-208. ISSN 0167-7012

Preview

PDF WRAP_Mills_Draft_JMM_InPress.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (369Kb)

Abstract

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System™, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi.

Item Type:	Journal Article
Subjects:	S Agriculture > S Agriculture (General)
Divisions:	Faculty of Science > Life Sciences (2010- ) > Warwick HRI (2004-2010)
Library of Congress Subject Headings (LCSH):	Coprinopsis cinerea, Genes
Journal or Publication Title:	Journal of Microbiological Methods
Publisher:	Elsevier
ISSN:	0167-7012
Date:	17 June 2008
Volume:	Vol.75
Number:	No.2
Page Range:	pp. 205-208
Identification Number:	10.1016/j.mimet.2008.06.006
Status:	Peer Reviewed
Access rights to Published version:	Open Access
Description:	Accepted manuscript.
References:	Binninger, D., Skrzynia, C., Pukkila, P., Casselton, L., 1987. DNA-mediated transformation of the basidiomycete Coprinopsis cinerea. EMBO J. 6, 835-840. Brummelkamp, T., Bernards, R., Agami, R., 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553. Challen, M.P., Kerrigan, R.W., Callac, P., 2003. A phylogenetic reconstruction and emendation of Agaricus section Duploannulatae. Mycologia 95, 61-73. Costa, A. M. S. B. (2007). Gene silencing strategies for functional genomics in homobasidiomycetes. PhD thesis, University of Bristol, 270 pp. De Jong, J.F., Deelstra, H.J., Wösten, H.A.B., Lugones, L.G., 2006. RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune. Appl. Environ. Microbiol. 72, 1267-1269. Goldoni, M., Azzalin, G., Macino, G., Cogoni, C., 2004. Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet. Biol. 41, 1016-1024. Granado, J.D., Kertesz-Chaloupková, K., Aebi, M., Kües, U., 1997. Restriction enzyme-mediated DNA Integration in Coprinopsis cinerea. Mol. Gen. Genet. 256, 28-36. Heneghan, M.N., Costa, A.M.S.B., Challen, M.P., Mills, P.R., Bailey A., Foster, G.D., 2007. A comparison of methods for successful triggering of gene silencing in Coprinopsis cinerea. Mol. Biotechnol. 35, 283-296. Kües, U., Klaus, M.J., Polak, E., Aebi, M., 2001. Multiple cotransformations in Coprinopsis cinerea. Fungal Genet. Newsl. 48, 32-34. Lewis, D., 1961. Genetic analysis of methionine suppressors in Coprinopsis. Genet. Res. 2, 141-155. Lu, S., Shi, R., Tsao, C., Yi, X., Li, L., Chiang, V., 2004. RNA silencing duplexes in plants by the expression of siRNA duplexes. Nucleic Acids Res. 32, e171. Namekawa, S.H., Iwabata, K., Sugawara, H., Hamada, F.N., Koshiyama, A., Chiku H., Kamada, T., Sakaguchi, K., 2005. Knockdown of LIM15/DMC1 in the mushroom Coprinopsis cinerea by double-stranded RNA-mediated gene silencing. Microbiology 151, 3669-3678. Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., Conklin, D.S., 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16, 948-958. Rappleye, C., Engle, J., Goldman, W., 2004. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol. Microbiol. 53, 153-165. Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P.S., Paddison, P.J., Hannon, G.J., Cleary, M.A., 2005 Synthetic shRNAs as potent RNAi triggers. Nature Biotechnol. 23, 227–231. Sreenivasaprasad, S., 2000. Isolation of fungal nucleic acids. In: Rapley, R. (Ed.), The Nucleic Acid Protocols Handbook. Humana Press, New Jersey, pp. 37-45. Tanguay, P., Bozza, S., Breuil, C., 2006. Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae. Fungal Genet. Biol. 43, 804-812. Wälti, M.A., Villalba, C., Buser, R.M., Grunler, A., Aebi, M., Künzler, M., 2006. Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression oh homologous hairpin RNAs. Eukaryot. Cell 5, 732-744. Yuan, J., Wang, X., Zhang, Y., Hu, X., Fei, J., Li, N., 2006. Mammalian Pol III promoter H1 can transcribe shRNA inducing RNAi in chicken cells. Mol. Biol. Rep. 33, 33-41.
URI:	http://wrap.warwick.ac.uk/id/eprint/191

Request changes to a record

Actions (login required)

View Item