PT  - JOURNAL ARTICLE
AU  - Manuela S. Pieperhoff
AU  - Gurman S. Pall
AU  - Elena Jiménez-Ruiz
AU  - Sujaan Das
AU  - Eleanor H Wong
AU  - Joanne Heng
AU  - Sylke Müller
AU  - Michael J Blackman
AU  - Markus Meissner
TI  - Conditional U1 Gene Silencing in <em>Toxoplasma gondii</em>
AID  - 10.1101/008649
DP  - 2014 Jan 01
TA  - bioRxiv
PG  - 008649
4099  - http://biorxiv.org/content/early/2014/09/01/008649.short
4100  - http://biorxiv.org/content/early/2014/09/01/008649.full
AB  - In absence of powerful siRNA approaches, 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 exonintron 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 one-step approach 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 in Ku80 knockout and RH T. gondii tachyzoites. 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 new approach. This new gene silencing tool kit allows protein tracking and functional studies simultaneously.