Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi.
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.


CONCLUSIONS
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.
Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA. dxu@uga.edu
Dan
D
Xu
Cecilia Pérez
CP
Brandán
Miguel Angel
MA
Basombrío
Rick L
RL
Tarleton
D000818:Animals; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics*