Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

Autores
Xu, Dan; Pérez Brandan, Cecilia María; Basombrío, Miguel Ángel Manuel; Tarleton, Rick L
Año de publicación
2009
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Xu, Dan. University of Georgia; Estados Unidos
Fil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Tarleton, Rick L. University of Georgia; Estados Unidos
Materia
KNOCKOUT STRATEGIES
T. CRUZI
CHAGAS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/36582
Acceder
id CONICETDig_a01a053fde764b224e085183eed99caf
oai_identifier_str oai:ri.conicet.gov.ar:11336/36582
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Evaluation of high efficiency gene knockout strategies for Trypanosoma cruziXu, DanPérez Brandan, Cecilia MaríaBasombrío, Miguel Ángel ManuelTarleton, Rick LKNOCKOUT STRATEGIEST. CRUZICHAGAShttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Background. 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.Fil: Xu, Dan. University of Georgia; Estados UnidosFil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Tarleton, Rick L. University of Georgia; Estados UnidosBioMed Central2009-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/36582Xu, Dan; Pérez Brandan, Cecilia María; Basombrío, Miguel Ángel Manuel; Tarleton, Rick L; Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi; BioMed Central; Bmc Microbiology; 9; 90; 5-2009; 90-1001471-2180CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1186/1471-2180-9-90info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:43:30Zoai:ri.conicet.gov.ar:11336/36582instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:43:30.947CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
title Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
spellingShingle Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
Xu, Dan
KNOCKOUT STRATEGIES
T. CRUZI
CHAGAS
title_short 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_sort Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
dc.creator.none.fl_str_mv Xu, Dan
Pérez Brandan, Cecilia María
Basombrío, Miguel Ángel Manuel
Tarleton, Rick L
author Xu, Dan
author_facet Xu, Dan
Pérez Brandan, Cecilia María
Basombrío, Miguel Ángel Manuel
Tarleton, Rick L
author_role author
author2 Pérez Brandan, Cecilia María
Basombrío, Miguel Ángel Manuel
Tarleton, Rick L
author2_role author
author
author
dc.subject.none.fl_str_mv KNOCKOUT STRATEGIES
T. CRUZI
CHAGAS
topic KNOCKOUT STRATEGIES
T. CRUZI
CHAGAS
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv 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.
Fil: Xu, Dan. University of Georgia; Estados Unidos
Fil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Tarleton, Rick L. University of Georgia; Estados Unidos
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.
publishDate 2009
dc.date.none.fl_str_mv 2009-05
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/36582
Xu, Dan; Pérez Brandan, Cecilia María; Basombrío, Miguel Ángel Manuel; Tarleton, Rick L; Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi; BioMed Central; Bmc Microbiology; 9; 90; 5-2009; 90-100
1471-2180
CONICET Digital
CONICET
url http://hdl.handle.net/11336/36582
identifier_str_mv Xu, Dan; Pérez Brandan, Cecilia María; Basombrío, Miguel Ángel Manuel; Tarleton, Rick L; Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi; BioMed Central; Bmc Microbiology; 9; 90; 5-2009; 90-100
1471-2180
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1186/1471-2180-9-90
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1844613369164726272
score 13.070432

Publicaciones similares