Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi.

Xu D, Brandܡn CP, Basombrܭo MA, Tarleton RL.
Source: BMC Mol Biol
Publication Date: (2009)
Issue: 11: 1-10
Nucleofector® I/II/2b
A total of 1 Ü? 107 early-log epimastigotes were centrifuged at 1,620 g for 15 min and resuspended in 100 μl room temperature Human T Cell Nucleofector™ Solution (Amaxa AG, Cologne, Germany). The resuspended parasites were then mixed with 3–10 μg DNA (8–10 μg DNA for constructs generated using the conventional and MS/GW strategy; 3–10 μg DNA for constructs generated through one-step-PCR) in a total volume of 5–10 μl and electroporated using program U-33 in an AMAXA Nucleofector Device. This protocol generally yields 6–13% yellow fluorescent protein (YFP) positive parasites 24 hrs after transfection using 10 μg of a YFP-containing control plasmid.
BACKGROUND: Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. RESULTS: While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes. CONCLUSION: Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi.