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: | Heng, Dr Joanne and Pall, Dr Gurman and Muller, Professor Sylke and Meissner, Professor Markus and Pieperhoff, Dr Manuela and Wong, Dr Eleanor and Das, Dr Sujaan and Jimenez Ruiz, Dr Elena 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 > School of Infection & Immunity |
| 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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