Gene tagging in Trypanosoma cruzi.

CANTALUPI, MARISOL; PERUGORIA, GONZALO; BOUVIER, LEÓN A.; NIEMIROWICZ, GABRIELA T.

La Plata

Congreso; XXXIV Reunión Anual de la Sociedad Argentina de Protozoología; 2023

Sociedad Argentina de Protozoología

Genome editing techniques are essential to understand different biological and cellular processes. Historically, genetic manipulation of Trypanosoma cruzi has been challenging compared to other pathogenic protozoans.Nowadays, most gene tagging strategies available rely on vectors that overexpress the protein of interest or on CRISPR/Cas9-based gene insertion methodologies. Both these options demand considerable time and technical expertise. Additionally, the use of pre-established T. cruzi cell lines are occasionally essential. To overcome these limitations, we devised a simple PCR-based strategy to target different chromosomal loci for the insertion of different epitope tags. To test this approach, we constructed a series of template plasmids that carry the mEGFP reporter and the neomycin or the blasticidin S resistance genes. These DNA segments were spliced by overlap extension PCR into a single molecule with homologous sections derived from the targeted gene, producing C-terminal fusions. To investigate the performance of this method, we generate an epimastigote cell line that constitutively expresses the mCherry fluorescent protein using the pROCK vector. This gene was interrupted with the mEGFP sequence employing genetic constructs with different recombination length. Using this strategy, after four weeks of selection, approximately 95% of the transgenic epimastigote population lost the expression of the mCherry protein. Cloned cell lines analysis by PCR followed by DNA sequencing indicated that the constructs had accurately integrated into the mCherry locus. Moreover, we were able to detect mEGFP protein expression by Western blot and flow cytometry analysis. In contrast to much more complex approaches, the strategies presented herein only require elemental and broadly available molecular biology techniques and constitute a cost and time-efficient alternative for the endogenous labeling of genes in T. cruzi.