title: Conditional U1 Gene Silencing in Toxoplasma gondii.
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.
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Manuela S
authors.initials: MS
authors.lastname: Pieperhoff
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Gurman S
authors.initials: GS
authors.lastname: Pall
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Elena
authors.initials: E
authors.lastname: Jiménez-Ruiz
authors.affiliation: Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.
authors.forename: Sujaan
authors.initials: S
authors.lastname: Das
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Carmen
authors.initials: C
authors.lastname: Melatti
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Gow
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Eleanor H
authors.initials: EH
authors.lastname: Wong
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Joanne
authors.initials: J
authors.lastname: Heng
authors.affiliation: Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Sylke
authors.initials: S
authors.lastname: Müller
authors.affiliation: Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow, Lanarkshire, United Kingdom.
authors.forename: Markus
authors.initials: M
authors.lastname: Meissner
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D033922:Clathrin Heavy Chains / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D020868:Gene Silencing; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D010963:Plasmodium falciparum / Q000235:genetics; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014122:Toxoplasma / Q000235:genetics*
chemical_list: D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear; D033922:Clathrin Heavy Chains