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Simplified inducible system for Trypanosoma brucei.
- 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.
- Subjects :
- Cell Line
Genetic Vectors
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
Protozoan Proteins genetics
Protozoan Proteins metabolism
RNA Interference
Transcriptional Activation
Genetic Techniques
Trypanosoma brucei brucei genetics
Trypanosoma brucei brucei metabolism
Subjects
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