Endogenous C-terminal tagging by PCR SOEing in Trypanosoma cruzi.

LEÓN ALBERTO BOUVIER; MARISOL CANTALUPI; GONZALO PERUGORRIA; GABRIELA TERESA NIEMIROWICZ

Buenos Aires

Congreso; XXXIII Reunión Anual de la Sociedad Argentina de Protozoología; 2022

High-throughput methods to establish the precise subcellular localization of endogenous proteins or to identify interaction partners within larger complexes are essential to understand different cellular processes. While specific antibodies are useful tools to solve these aspects, gene tagging approaches have become popular, allowing the analysis of diverse proteins in realistic timeframes. For Trypanosoma cruzi, gene tagging strategies rely on vectors that overexpress the protein of interest or alternatively on CRISPR/Cas9 based gene insertion methodologies. Both these options are time-consuming and technically challenging. Furthermore, occasionally pre-established cell lines are an indispensable requirement. To overcome these limitations, we devised a simple PCR-based strategy to target different chromosomal loci for insertion of GFP and epitope tags. To test this approach, we constructed a series of template plasmids that carry the eGFP 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. The utility of the method was established by tagging genes encoding proteins of known localization such as the paraflagellar rod protein 2 (PFR2); Bilbo 1, a component of the flagellar pocket collar and TcMCP-1, a cytosolic metallocarboxypeptidase. After four weeks of selection, this methodology proved to be highly efficient (> 95%) in all recombinant cell lines generated. Moreover, as determined by Western blot analysis, for TcMCP-1 we were able to endogenously replace both native alleles with tagged derivatives. 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 labelling of genes in T. cruzi.