Workshop: Alternative approaches for control of gene expression in trypanosomatids.

NIEMIROWICZ, GABRIELA T.; ALVAREZ, VANINA E.; BOUVIER, LEÓN A.

Mar del Plata

Congreso; XXXI Reunión Anual de la Sociedad Argentina de Protozoología (SAP); 2019

Sociedad Argentina de Protozoología (SAP)

Regulatable gene expression in trypanosomatids relies on pre-establishing cell lines modified to express exogenous regulatory elements such as bacterial repressors and polymerases and therefore, inducible experimentation is frequently restricted to few laboratory strains. Moreover, extending these methodologies to other strains usually requires several additional transfection steps as well as lengthy periods in culture thus affecting the natural wild type properties sought after in field isolates.Here we explored two alternative simplified strategies for inducible transgene expression in Trypanosoma cruzi and Trypanosoma brucei. First we studied the drug dependent modulation of GFP mRNA stability by insertion of a cis-acting riboswitch. This element consists of a Schistosoma mansoni hammerhead ribozyme fused to a tetracycline aptamer. Reporter GFP expression could be downregulated by tetracycline addition, in T. cruzi epimastigotes as well as in procyclic T. brucei trypomastigotes. Alternatively, for the later model organism, we established a simplified inducible expression system dependent exclusively on RNA polymerase I transcription. The fewer exogenous regulatory elements involved, made possible to accommodate the entire system along with the sequence of interest on a single vector. Consequently, the obtention of an inducible cell line could be accomplished, in a single step, by transfection into any wild type procyclic or bloodstream form cell line. This tool not only allowed inducible expression of GFP, but it also proved useful for the RNAi based down regulation of essential genes, such as those for alfa-tubulin, enolase and clathrin heavy chain. Equivalent vectors are currently being developed for Trypanosoma cruzi.