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dc.contributor.authorXu, Dan
dc.contributor.authorPérez Brandán, Cecilia
dc.contributor.authorBasombrío, Miguel Á
dc.contributor.authorTarleton, Rick L
dc.date.accessioned2013-06-12T15:12:09Z
dc.date.available2013-06-12T15:12:09Z
dc.date.issued2009-05-11
dc.identifier.citationBMC Microbiology. 2009 May 11;9(1):90
dc.identifier.urihttp://dx.doi.org/10.1186/1471-2180-9-90
dc.identifier.urihttp://hdl.handle.net/10724/19729
dc.description.abstractAbstract 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.
dc.titleEvaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
dc.typeJournal Article
dc.date.updated2013-06-07T18:23:03Z
dc.description.versionPeer Reviewed
dc.language.rfc3066en
dc.rights.holderDan Xu et al.; licensee BioMed Central Ltd.


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