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

Abstract

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes ofinterest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will onlyguarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selectionwith these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage,we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. Totest this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance(Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A)was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasitepopulations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediatedfusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenoustagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene.The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgeneexpression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenouslabeling of genes.