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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 experim...

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Autores principales: Xu, Dan, Pérez Brandán, Cecilia, Basombrío, Miguel Ángel, Tarleton, Rick L
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688506/
https://www.ncbi.nlm.nih.gov/pubmed/19432966
http://dx.doi.org/10.1186/1471-2180-9-90
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author Xu, Dan
Pérez Brandán, Cecilia
Basombrío, Miguel Ángel
Tarleton, Rick L
author_facet Xu, Dan
Pérez Brandán, Cecilia
Basombrío, Miguel Ángel
Tarleton, Rick L
author_sort Xu, Dan
collection PubMed
description 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.
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spelling pubmed-26885062009-05-30 Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi Xu, Dan Pérez Brandán, Cecilia Basombrío, Miguel Ángel Tarleton, Rick L BMC Microbiol Methodology article 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. BioMed Central 2009-05-11 /pmc/articles/PMC2688506/ /pubmed/19432966 http://dx.doi.org/10.1186/1471-2180-9-90 Text en Copyright ©2009 Xu et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology article
Xu, Dan
Pérez Brandán, Cecilia
Basombrío, Miguel Ángel
Tarleton, Rick L
Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title_full Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title_fullStr Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title_full_unstemmed Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title_short Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title_sort evaluation of high efficiency gene knockout strategies for trypanosoma cruzi
topic Methodology article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688506/
https://www.ncbi.nlm.nih.gov/pubmed/19432966
http://dx.doi.org/10.1186/1471-2180-9-90
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