title: Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters.
abstract: The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.
authors.affiliation: Department of Biochemistry, College of Medicine, 4-403 Bowen Science Building, 51 Newton Road, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: LaCount
authors.forename: S
authors.initials: S
authors.lastname: Bruse
authors.forename: K L
authors.initials: KL
authors.lastname: Hill
authors.forename: J E
authors.initials: JE
authors.lastname: Donelson
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D020868:Gene Silencing; D017125:Genes, Protozoan; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D006657:Histones; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D012330:RNA, Double-Stranded; D016053:RNA, Protozoan; D014404:Tubulin; C045519:paraflagellar rod proteins, Trypanosoma brucei; D049452:Green Fluorescent Proteins