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Simplified inducible system for Trypanosoma brucei.

Authors :
Niemirowicz GT
Cazzulo JJ
Álvarez VE
Bouvier LA
Source :
PloS one [PLoS One] 2018 Oct 11; Vol. 13 (10), pp. e0205527. Date of Electronic Publication: 2018 Oct 11 (Print Publication: 2018).
Publication Year :
2018

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.<br />Competing Interests: The authors have declared that no competing interests exist.

Details

Language :
English
ISSN :
1932-6203
Volume :
13
Issue :
10
Database :
MEDLINE
Journal :
PloS one
Publication Type :
Academic Journal
Accession number :
30308039
Full Text :
https://doi.org/10.1371/journal.pone.0205527