Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi

Autores
Niemirowicz, Gabriela Teresa; Carlevaro, Giannina Alejandra; Campetella, Oscar Eduardo; Bouvier, Leon Alberto; Mucci, Juan Sebastián
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes ofinterest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will onlyguarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selectionwith these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage,we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. Totest this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance(Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A)was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasitepopulations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediatedfusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenoustagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene.The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgeneexpression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenouslabeling of genes.
Fil: Niemirowicz, Gabriela Teresa. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Carlevaro, Giannina Alejandra. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Bouvier, Leon Alberto. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Mucci, Juan Sebastián. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Materia
CHAGAS DISEASE
GENE EXPRESSION
EXPRESSION VECTORS
TRYPANOSOMA CRUZI
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/265940
Acceder
id CONICETDig_1c0850cbc6c7158bf66336776097e02c
oai_identifier_str oai:ri.conicet.gov.ar:11336/265940
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruziNiemirowicz, Gabriela TeresaCarlevaro, Giannina AlejandraCampetella, Oscar EduardoBouvier, Leon AlbertoMucci, Juan SebastiánCHAGAS DISEASEGENE EXPRESSIONEXPRESSION VECTORSTRYPANOSOMA CRUZIhttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes ofinterest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will onlyguarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selectionwith these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage,we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. Totest this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance(Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A)was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasitepopulations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediatedfusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenoustagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene.The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgeneexpression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenouslabeling of genes.Fil: Niemirowicz, Gabriela Teresa. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carlevaro, Giannina Alejandra. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Bouvier, Leon Alberto. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Mucci, Juan Sebastián. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaElsevier2024-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/265940Niemirowicz, Gabriela Teresa; Carlevaro, Giannina Alejandra; Campetella, Oscar Eduardo; Bouvier, Leon Alberto; Mucci, Juan Sebastián; Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi; Elsevier; Heliyon; 10; 2; 1-2024; 1-132405-8440CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.heliyon.2024.e24595info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:11:23Zoai:ri.conicet.gov.ar:11336/265940instacron: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:34982025-09-10 13:11:24.098CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
title Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
spellingShingle Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
Niemirowicz, Gabriela Teresa
CHAGAS DISEASE
GENE EXPRESSION
EXPRESSION VECTORS
TRYPANOSOMA CRUZI
title_short Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
title_full Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
title_fullStr Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
title_full_unstemmed Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
title_sort Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi
dc.creator.none.fl_str_mv Niemirowicz, Gabriela Teresa
Carlevaro, Giannina Alejandra
Campetella, Oscar Eduardo
Bouvier, Leon Alberto
Mucci, Juan Sebastián
author Niemirowicz, Gabriela Teresa
author_facet Niemirowicz, Gabriela Teresa
Carlevaro, Giannina Alejandra
Campetella, Oscar Eduardo
Bouvier, Leon Alberto
Mucci, Juan Sebastián
author_role author
author2 Carlevaro, Giannina Alejandra
Campetella, Oscar Eduardo
Bouvier, Leon Alberto
Mucci, Juan Sebastián
author2_role author
author
author
author
dc.subject.none.fl_str_mv CHAGAS DISEASE
GENE EXPRESSION
EXPRESSION VECTORS
TRYPANOSOMA CRUZI
topic CHAGAS DISEASE
GENE EXPRESSION
EXPRESSION VECTORS
TRYPANOSOMA CRUZI
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes ofinterest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will onlyguarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selectionwith these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage,we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. Totest this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance(Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A)was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasitepopulations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediatedfusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenoustagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene.The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgeneexpression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenouslabeling of genes.
Fil: Niemirowicz, Gabriela Teresa. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Carlevaro, Giannina Alejandra. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Campetella, Oscar Eduardo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Bouvier, Leon Alberto. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Mucci, Juan Sebastián. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina
description Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes ofinterest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will onlyguarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selectionwith these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage,we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. Totest this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance(Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A)was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasitepopulations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediatedfusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenoustagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene.The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgeneexpression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenouslabeling of genes.
publishDate 2024
dc.date.none.fl_str_mv 2024-01
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/11336/265940
Niemirowicz, Gabriela Teresa; Carlevaro, Giannina Alejandra; Campetella, Oscar Eduardo; Bouvier, Leon Alberto; Mucci, Juan Sebastián; Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi; Elsevier; Heliyon; 10; 2; 1-2024; 1-13
2405-8440
CONICET Digital
CONICET
url http://hdl.handle.net/11336/265940
identifier_str_mv Niemirowicz, Gabriela Teresa; Carlevaro, Giannina Alejandra; Campetella, Oscar Eduardo; Bouvier, Leon Alberto; Mucci, Juan Sebastián; Monocistronic expression system and endogenous gene labeling through the T2A strategy in Trypanosoma cruzi; Elsevier; Heliyon; 10; 2; 1-2024; 1-13
2405-8440
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.heliyon.2024.e24595
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
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
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score 12.993085

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