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Mol Biochem Parasitol. 2008 Sep;161(1):76-9. doi: 10.1016/j.molbiopara.2008.05.006. Epub 2008 Jun 7.

Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei.

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London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.


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.

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