Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi

• A rapid method for transgene expression in intracellular T. cruzi amastigotes.
• Direct transfection of trypomastigotes prior to host cell infection.
• FACS sorting of transfected trypomastigotes is an effective enrichment tool.
• Method by-passes standard epimastigote transfection and selection steps.

The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field.

Figure optionsDownload high-quality image (55 K)Download as PowerPoint slide