Development of high-throughput gene knockout strategies in Trypanosoma cruzi for production of attenuated lines
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In contrast to the substantial in silico studies of the T. cruzi genome, only a few genes have been experimentally characterized, mainly due to the lack of convenient methods for gene manipulation for reverse genetics studies. 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. 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 delete target genes. Using this strategy, we have successfully generated single- and double- gene knockout parasites with genes that are predicted to be non-essential for epimastigote survival but essential for growth of T. cruzi amastigotes.