Conditional U1 Gene Silencing in <i>Toxoplasma gondii</i>

<div><p>The functional characterisation of essential genes in apicomplexan parasites, such as <i>Toxoplasma gondii</i> or <i>Plasmodium falciparum</i>, 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 <i>T</i>. <i>gondii</i> including the clathrin heavy chain gene 1 <i>(chc1)</i>, the vacuolar protein sorting gene 26 (<i>vps26)</i>, and the dynamin-related protein C gene (<i>drpC)</i> 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.</p>