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

Sam Alsford, David Horn

    Research output: Contribution to journalArticlepeer-review

    85 Citations (Scopus)

    Abstract

    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. (C) 2008 Elsevier B.V. All rights reserved.

    Original languageEnglish
    Pages (from-to)76-79
    Number of pages4
    JournalMolecular and Biochemical Parasitology
    Volume161
    Issue number1
    DOIs
    Publication statusPublished - Sep 2008

    Keywords

    • GENE-EXPRESSION
    • AFRICAN TRYPANOSOME
    • SYSTEM
    • EFFICIENCY
    • epigenetic
    • DOUBLE-STRANDED-RNA
    • position-effects
    • GENOME
    • functional-genomics
    • RNAi
    • localisation
    • INTERFERENCE
    • SELECTION

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