Development of high-throughput gene knockout strategies in Trypanosoma cruzi for production of attenuated lines

DAN XU, CECILIA PÉREZ BRANDAN, RICK L. TARLETON

Philadelphia, USA

Conferencia; American Society of Tropical Medicine and Hygiene 56th Annual Meeting.; 2007

Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease in humans, infects approximately 16-18 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, mainly due to the lack of convenient methods for gene manipulation for reverse genetics studies. Current strategies for gene disruption in T. cruzi are tedious and a quick and easy strategy to knockout genes is needed. 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. Our results show that while the one-step-PCR strategy is faster than the other methods, it does not efficiently target genes of interest and gene-specific sequences of 78 nucleotides are not sufficient to guarantee homologous recombination. The Multisite Gateway based approach, although not as fast and easy as the one-step-PCR strategy, is less time consuming than the conventional method and is able to efficiently disrupt target genes. Using the Multisite Gateway strategy, we have successfully generated parasites deleted of several genes that are predicted to be non-essential for epimastigote survival but essential for growth of T. cruzi amastigotes. Those knockout parasites are currently being screened for their ability to persist in mice. The results of this study will not only facilitate reverse genetic studies of T. cruzi genes, but also will facilitate the development of live attenuated parasites to be tested as experimental vaccines for Chagas disease.