Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei.
A major obstacle to reproducible expression of recombinant transcripts lies in the epigenetic effects of the flanking chromatin following integration. We previously presented a strategy to overcome this problem in bloodstream form Trypanosoma brucei, using a reporter to identify a ribosomal-spacer locus that supports optimal expression and then marking that locus for subsequent targeting. Advantages include elimination of variable-expression position-effects and the easy confirmation of correct integration. We now report a set of validated constructs that exploit this system for expression of dsRNA or recombinant protein. The current construct-set allows expression of intramolecular dsRNA for RNA interference knockdown or expression of proteins that can incorporate c-Myc epitope(s) or a fluorescent-tag for subcellular localisation, interaction and/or other functional analysis. The constructs are integrated at a single, marked locus and deliver reliable and reproducible expression.
Alsford
Sam
S
London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
Horn
David
D
D000818:Animals; D015870:Gene Expression; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D012330:RNA, Double-Stranded / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D019076:Transgenes; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D012330:RNA, Double-Stranded; D011994:Recombinant Proteins