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Simplified inducible system for Trypanosoma brucei.
Gabriela T Niemirowicz1, Juan J Cazzulo1, Vanina E Álvarez1
1Instituto de Investigaciones Biotecnológicas (IIB) Dr. Rodolfo A. Ugalde, Universidad Nacional de San Martín (UNSAM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Buenos Aires, Argentina.
Plos One
|October 12, 2018
Summary
Researchers developed a new system for Trypanosoma brucei gene expression and RNA interference. This adaptable tool works across different strains, enabling new research possibilities for this important parasite.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Current reverse genetics in Trypanosoma brucei are limited to specific cell lines, restricting inducible experimentation.
- A need exists for adaptable genetic tools applicable to diverse Trypanosoma brucei strains.
Purpose of the Study:
- To develop a novel, adaptable transgene expression and RNA interference system for Trypanosoma brucei.
- To enable inducible gene manipulation in various Trypanosoma brucei forms and strains.
Main Methods:
- Designed pTbFIX vectors utilizing endogenous rRNA promoters and a tetracycline-inducible system.
- Incorporated nucleus-targeted tetracycline repressor and puromycin resistance genes.
- Validated the system for both GFP expression and RNA interference (RNAi) in bloodstream and procyclic forms.
Main Results:
- Achieved robust, doxycycline-inducible GFP expression (170-400 fold) in both T. brucei forms.
- Successfully recapitulated lethal phenotypes using RNAi targeting α-tubulin, enolase, and clathrin heavy chain genes.
- Demonstrated the system's adaptability across different T. brucei strains.
Conclusions:
- The pTbFIX system offers a versatile and adaptable platform for reverse genetics in Trypanosoma brucei.
- This new tool expands the scope of inducible experimentation and RNAi in this medically and veterinarily important protozoan parasite.
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