title: Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects.
abstract: In Trypanosoma brucei, RNA interference (RNAi) and recombinant protein expression are established as powerful approaches for functional genomics, particularly when combined with inducible expression. The favoured methods involve exploiting homologous recombination to target expression cassettes to a chromosome sub-set to establish stable cell lines. Unfortunately, bloodstream-form cells, those that cause disease in mammals, exhibit low efficiency stable transfection. Current expression systems can also exhibit other undesirable features, including variable position effects and leaky, inducible expression. We have developed systems in bloodstream-form cells that alleviate these problems. Using constructs for RNAi and expression of (GFP) tagged proteins, we target a (hyg) tagged ribosomal RNA (RRNA) locus which circumvents position effects and allows increased targeting efficiency. We also report a compatible double-inducible system for tight regulation of highly toxic products. This system exploits a new inducible RRNA promoter to drive T7 RNA polymerase (T7RNAP) transcription which then drives expression from inducible T7 promoters. The developments described should facilitate functional analysis and increased throughput.
authors.affiliation: Department of Infections and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Alsford
authors.forename: Taemi
authors.initials: T
authors.lastname: Kawahara
authors.forename: Lucy
authors.initials: L
authors.lastname: Glover
authors.forename: David
authors.initials: D
authors.lastname: Horn
mesh_terms: D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D016053:RNA, Protozoan / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases