Development of simplified inducible expression systems for trypanosomatids

NIEMIROWICZ, GABRIELA TERESA; CAZZULO, JUAN JOSÉ; ALVAREZ, VANINA EDER; BOUVIER, LEÓN ALBERTO

Buenos Aires

Congreso; Reunión Conjunta de las Sociedades de Biociencias; 2017

In trypanosomatids, inducible expression systems are based on the sequential use of two types of genetic manipulation vectors. First, wild type strains have to be transfected with plasmids that code for regulatory elements such as bacterial repressors and polymerases. It is only after establishing these cell lines that is possible to modulate the expression of sequences of interest carried in the second type of plasmid that make up the system. In addition, since the discovery of highly processive RNA transcription of bacteriophage polymerases in T. brucei, these enzymes have been included in almost all inducible expression systems developed so far, although endogenous promoters that allow constitutive as well as regulatable expression have been described.In this work we conceived a simplified inducible expression system based entirely on endogenous promoters with operators for bacterial repressors. It is worth noting that along with reporter genes and selectable markers the latter represent the only included foreign elements. This reduced set, made possible to accommodate the entire system together with the sequence of interest in a single vector and thus, the obtention of an inducible cell line can be accomplished by one step transfection of a wild type strain.Due to the fact that T. brucei is more amenable to genetic manipulation, initial prototypes were designed for this organism and transfected in procyclic Lister-427 trypomastigotes. After selection, Western blot analysis revealed a 35-fold expression induction of the reported GFP gene compared to non-induced cells.The system described here will represent a useful molecular genetic tool that, independently of the strain background, will enable tightly-regulated protein expression and stem-loop based RNAi strategies. Once established, it will constitute the starting point for the development of equivalent systems for other trypanosomatids.