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

Article English OPEN
Costa, Ana M.S.B. ; Mills, P. R. ; Bailey, Andy M. ; Foster, Gary D. ; Challen, Michael P. (2008)

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.\ud \ud
  • References (13)
    13 references, page 1 of 2

    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 Tsilencing by expression of double stranded RNA in Neurospora crassa. FIuPngal Genet. Biol. 41, 1016-1024. R

    Granado, J.D., Kertesz-Chaloupková, K., Aebi, M., Kües, UC.,1997. Restriction enzyme-mediated DNA Integration in Coprinopsis Scinerea. Mol. Gen. Genet. 256, 28-36. NU

    Heneghan, M.N., Costa, A.M.S.B., Challen, MA.P., Mills, P.R., Bailey A., Foster, G.D., 2007. A comparison of methods Mfor successful triggering of gene silencing in Coprinopsis cinerea. Mol. Biotechnol. 35, 283-296. D

    Kües, U., Klaus, M.J., Polak, E., AEebi, M., 2001. Multiple cotransformations in Coprinopsis cinerea. FunTgal Genet. Newsl. 48, 32-34. P

    Lewis, D., 1961. Genetic analysis of methionine suppressors in Coprinopsis. Genet. Res. 2, 141-155C.E

    Lu, S., Shi, R., TsCao,C., Yi, X., Li, L., Chiang, V., 2004. RNA silencing duplexes in plants by Athe 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

  • Metrics
    views in OpenAIRE
    views in local repository
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Warwick Research Archives Portal Repository - IRUS-UK 0 89
Share - Bookmark