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.
Highlights
Protozoan parasites of the phylum Apicomplexa, such as Toxoplasma gondii, Plasmodium spp. or Cryptosporidium spp., are important pathogens of human and livestock populations
Since the spliceosome and the mechanisms involved in the definition of exon-intron boundaries are highly conserved in eukaryotes [10], we reasoned that positioning U1 recognition sequences in the terminal exon of a T. gondii genes of interest (GOI) would result in efficient gene knockdown, as previously shown in other eukaryotes [16]
Polyadenylation and consequent degradation of the pre-mRNA, resulting in an effective knockdown of expression levels of a GOI (Fig 1B). To test whether this method can be applied in T. gondii, based on application experience in mammalian systems with multiple binding sites increasing the level of inhibition in a synergistic fashion [17] two U1 recognition sequences were positioned in tandem directly downstream of the STOP codon of the reporter gene mycGFP
Summary
Protozoan parasites of the phylum Apicomplexa, such as Toxoplasma gondii, Plasmodium spp. or Cryptosporidium spp., are important pathogens of human and livestock populations. Highthroughput analyses of essential genes of interest (GOI) are lacking since current reverse genetic technologies are time consuming [1].
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