Abstract
Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including α-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.