Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi

CARLEVARO G; MUCCI J; BOUVIER L; NIEMIROWICZ G

Mar del Plata, Buenos Aires

Congreso; Reunión anual de sociedades de biociencia-XXXI reunión anual de la sociedad argentina de protozoología; 2019

To date, most gene expression systems available for T. cruzi are based on policistronic vectors. In these, messenger RNAs for both, the gene of interest and the selectable marker, mature independently from the same primary transcript. Due to this independence, a common disadvantage of this configuration is the selection of resistant cells lacking expression of the gene of interest. In this sense, the selective agent only assures the expression of the resistance gene. To overcome this limitation, we devised a monocistronic vector in which the gene of interest and the selectable marker are coded on the same mRNA linked in frame by the sequence for a 2A peptide. To test this approach, we introduced the Thosea asigna virus self-cleaving 2A peptide between the eGFP reporter and the neomycin resistance gene. For comparative purposes we derived an additional plasmid with a bicistronic configuration by insertion of a GAPDH intergenic sequence in place of that for the 2A and transfected three different strains of T. cruzi. Flow cytometry analysis showed higher eGFP expression levels and more homogeneous populations when comparing cells obtained with the monocistronic vector than those transfected with the bicistronic one. Moreover, fusion protein dissociation occurred with high efficiency (>94%) as determined by Western blot. In addition to that, this system is highly stable and is active in all stages of the parasite. Given the resulting efficiency, we successfully adapted the monocistronic vector to easily introduce a hemagglutinin tag to the actin gene by ends-in homologous recombination. In conclusion, the use of ribosome skipping peptides in the context of a monocistronic expression vector, can be used as a cost efficient alternative not only to improve protein expression in T. cruzi, but also to label endogenous genes without needing complex systems, such as the CRISPR/Cas9.