Rna interference in cryptococcus neoformans

Michael L. Skowyra, Tamara L. Doering

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

14 Scopus citations

Abstract

RNA interference (RNAi) is an experimental technique used to suppress individual gene expression in eukaryotic cells in a sequence-dependent manner. The process relies on double-stranded RNA (dsRNA) to target complementary messenger RNA for degradation. Here, we describe two plasmid-based strategies we have developed for RNAi in Cryptococcus neoformans. The pFrame vector utilizes the ACT1 promoter to enable the constitutive synthesis of hairpin RNA (hpRNA), the stem of which constitutes the dsRNA trigger. The pIBB103 vector relies on convergent, inducible GAL7 promoters to independently drive the synthesis of the sense and antisense strands of the interfering sequence; these strands anneal to form the initiating dsRNA molecule. Both vectors are designed to co-silence a “sentinel” gene with an easily scored phenotype to help identify clones in which RNAi is most effective. We provide guidelines for selecting a suitable interfering sequence to trigger RNAi in C. neoformans and describe the steps for subcloning into either vector, transforming C. neoformans by electroporation, screening clones for RNAi-related phenotypes, and evaluating the efficacy and specificity of gene silencing by RNAi.

Original languageEnglish
Title of host publicationHost-Fungus Interactions
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages165-186
Number of pages22
ISBN (Print)9781617795381
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume845
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Cryptococcus neoformans
  • Gene expression
  • Gene silencing
  • RNA interference

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