Simplified inducible system for Trypanosoma brucei

Autores
Niemirowicz, Gabriela Teresa; Cazzulo, Juan Jose; Alvarez, Vanina Eder; Bouvier, León Alberto
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including a-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.
Fil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Bouvier, León Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Materia
Trypanosoma
Cloning
RNA interference
Ribosomal RNA
Plasmid construction
Transcriptional control
Gene expression
Trypanosoma
Ribosomal RNA
Plasmid construction
Transcriptional control
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:11336/96969
Acceder
id CONICETDig_1a833ae10222f64fdee25bee0c7f6946
oai_identifier_str oai:ri.conicet.gov.ar:11336/96969
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Simplified inducible system for Trypanosoma bruceiNiemirowicz, Gabriela TeresaCazzulo, Juan JoseAlvarez, Vanina EderBouvier, León AlbertoTrypanosomaCloningRNA interferenceRibosomal RNAPlasmid constructionTranscriptional controlGene expressionTrypanosomaRibosomal RNAPlasmid constructionTranscriptional controlhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including a-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.Fil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Bouvier, León Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaPublic Library of Science2018-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/96969Niemirowicz, Gabriela Teresa; Cazzulo, Juan Jose; Alvarez, Vanina Eder; Bouvier, León Alberto; Simplified inducible system for Trypanosoma brucei; Public Library of Science; Plos One; 13; 10; 10-2018; 1-17; e02055271932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0205527info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205527info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:56:27Zoai:ri.conicet.gov.ar:11336/96969instacron: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:34982025-10-15 14:56:27.299CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Simplified inducible system for Trypanosoma brucei
title Simplified inducible system for Trypanosoma brucei
spellingShingle Simplified inducible system for Trypanosoma brucei
Niemirowicz, Gabriela Teresa
Trypanosoma
Cloning
RNA interference
Ribosomal RNA
Plasmid construction
Transcriptional control
Gene expression
Trypanosoma
Ribosomal RNA
Plasmid construction
Transcriptional control
title_short Simplified inducible system for Trypanosoma brucei
title_full Simplified inducible system for Trypanosoma brucei
title_fullStr Simplified inducible system for Trypanosoma brucei
title_full_unstemmed Simplified inducible system for Trypanosoma brucei
title_sort Simplified inducible system for Trypanosoma brucei
dc.creator.none.fl_str_mv Niemirowicz, Gabriela Teresa
Cazzulo, Juan Jose
Alvarez, Vanina Eder
Bouvier, León Alberto
author Niemirowicz, Gabriela Teresa
author_facet Niemirowicz, Gabriela Teresa
Cazzulo, Juan Jose
Alvarez, Vanina Eder
Bouvier, León Alberto
author_role author
author2 Cazzulo, Juan Jose
Alvarez, Vanina Eder
Bouvier, León Alberto
author2_role author
author
author
dc.subject.none.fl_str_mv Trypanosoma
Cloning
RNA interference
Ribosomal RNA
Plasmid construction
Transcriptional control
Gene expression
Trypanosoma
Ribosomal RNA
Plasmid construction
Transcriptional control
topic Trypanosoma
Cloning
RNA interference
Ribosomal RNA
Plasmid construction
Transcriptional control
Gene expression
Trypanosoma
Ribosomal RNA
Plasmid construction
Transcriptional control
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including a-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.
Fil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Fil: Bouvier, León Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
description Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including a-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.
publishDate 2018
dc.date.none.fl_str_mv 2018-10
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/11336/96969
Niemirowicz, Gabriela Teresa; Cazzulo, Juan Jose; Alvarez, Vanina Eder; Bouvier, León Alberto; Simplified inducible system for Trypanosoma brucei; Public Library of Science; Plos One; 13; 10; 10-2018; 1-17; e0205527
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/96969
identifier_str_mv Niemirowicz, Gabriela Teresa; Cazzulo, Juan Jose; Alvarez, Vanina Eder; Bouvier, León Alberto; Simplified inducible system for Trypanosoma brucei; Public Library of Science; Plos One; 13; 10; 10-2018; 1-17; e0205527
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0205527
info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205527
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
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
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_ 1846083099899723776
score 13.22299

Publicaciones similares