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BMC Microbiology

, 9:90

First Online: 11 May 2009Received: 18 December 2008Accepted: 11 May 2009

Abstract

BackgroundTrypanosoma 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.

ResultsWhile 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.

ConclusionUsing 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2180-9-90 contains supplementary material, which is available to authorized users.

Dan Xu, Cecilia Pérez Brandán contributed equally to this work.

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Autor: Dan Xu - Cecilia Pérez Brandán - Miguel Ángel Basombrío - Rick L Tarleton

Fuente: https://link.springer.com/article/10.1186/1471-2180-9-90







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