Functional genomics in Trypanosoma brucei: A collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci

The expression of transgenes encoding proteins modified o contain residues that impart a particular property, ‘tagged roteins’, is central to the post-genomic analysis of any organsm. Trypanosoma brucei is a model kinetoplastid protozoan athogen and has the most advanced repertoire of tools for everse genetic analysis available for any protozoan. The vast ajority of these tools take advantage of the predominance f homologous over non-homologous recombination to target onstructs to specific genomic loci. Initially the targeting was sed to direct unregulated transgenes to transcribed regions f the genome [1] and to perform gene deletions [2,3]. A eap forward in the sophistication of reverse genetic experments occurred with the development of trypanosome cell ines expressing the tetracycline repressor (TetR) protein which acilitated tetracycline-regulated expression of transgenes [4,5]. urther development of cell lines expressing both TetR and baceriophage T7 RNA polymerase (T7RNAP) allowed transgenes o be transcribed and expressed at very high levels [6]. The etRand T7RNAP-expressing cell lines are also central to most NA interference-based analyses of gene function currently erformed in trypanosomes [7–10]. In nearly all cases, an antibitic resistance gene is used as the selectable marker. The DNA sed for targetted integration is usually a linearised plasmid; argetting using PCR products directly is possible [11–13] but ntegration is less efficient and does not usually offer inducible xpression. The expression of tagged proteins has become central to the echnologies that have developed to analyse the function of indi-