Conditional U1 gene silencing in Toxoplasma gondii

Pieperhoff, M. et al. (2015) Conditional U1 gene silencing in Toxoplasma gondii. PLoS ONE, 10(6), e0130356. (doi:10.1371/journal.pone.0130356) (PMID:26090798) (PMCID:PMC4474610)

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Abstract

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Muller, Professor Sylke and Meissner, Professor Markus and Pieperhoff, Dr Manuela and Wong, Dr Eleanor and Jimenez Ruiz, Dr Elena and Heng, Dr Joanne and Pall, Dr Gurman and Das, Dr Sujaan and Gow, Mr Matthew
Authors: Pieperhoff, M., Pall, G., Jimenez Ruiz, E., Das, S., Melatti, C., Gow, M., Wong, E., Heng, J., Muller, S., Blackman, M. J., and Meissner, M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN:1932-6203
ISSN (Online):1932-6203
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in PLoS One 10(6):e0130356
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
501601Systematic analysis of essential parasite genes linked to invasion of the host cell in Toxoplasma gondiiMarkus MeissnerWellcome Trust (WELLCOME)087582/Z/08/ZIII - PARASITOLOGY
501602Systematic analysis of essential parasite genes linked to invasion of the host cell in Toxoplasma gondiiMarkus MeissnerWellcome Trust (WELLCOME)087582/Z/08/ZIII - PARASITOLOGY
371796The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)085349/Z/08/ZIII - PARASITOLOGY
371798The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)085349/B/08/ZIII - PARASITOLOGY