Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols
To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase e...
Guardado en:
Autores principales: | , , , , , , , , |
---|---|
Formato: | JOUR |
Materias: | |
Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_09320113_v92_n2_p113_DaRocha |
Aporte de: |
id |
todo:paper_09320113_v92_n2_p113_DaRocha |
---|---|
record_format |
dspace |
spelling |
todo:paper_09320113_v92_n2_p113_DaRocha2023-10-03T15:48:21Z Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols DaRocha, W.D. Silva, R.A. Bartholomeu, D.C. Pires, S.F. Freitas, J.M. Macedo, A.M. Vazquez, M.P. Levin, M.J. Teixeira, S.M.R. alpha tubulin beta tubulin DNA fragment immunoglobulin luciferase red fluorescent protein ribosome protein 5' untranslated region article electroporation epimastigote expression vector gene expression microbial genetics nonhuman priority journal reporter gene Trypanosoma cruzi 5' Untranslated Regions Animals Base Sequence Electroporation Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luciferases Luminescent Proteins Molecular Sequence Data Protozoan Proteins Transfection Trypanosoma cruzi Tubulin Protozoa Trypanosoma Trypanosoma cruzi To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5′ untranslated plus intergenic (5′ UTR plus Ig) regions from GAPDH, TcP2β, α- and β-tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5′ UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_09320113_v92_n2_p113_DaRocha |
institution |
Universidad de Buenos Aires |
institution_str |
I-28 |
repository_str |
R-134 |
collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
topic |
alpha tubulin beta tubulin DNA fragment immunoglobulin luciferase red fluorescent protein ribosome protein 5' untranslated region article electroporation epimastigote expression vector gene expression microbial genetics nonhuman priority journal reporter gene Trypanosoma cruzi 5' Untranslated Regions Animals Base Sequence Electroporation Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luciferases Luminescent Proteins Molecular Sequence Data Protozoan Proteins Transfection Trypanosoma cruzi Tubulin Protozoa Trypanosoma Trypanosoma cruzi |
spellingShingle |
alpha tubulin beta tubulin DNA fragment immunoglobulin luciferase red fluorescent protein ribosome protein 5' untranslated region article electroporation epimastigote expression vector gene expression microbial genetics nonhuman priority journal reporter gene Trypanosoma cruzi 5' Untranslated Regions Animals Base Sequence Electroporation Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luciferases Luminescent Proteins Molecular Sequence Data Protozoan Proteins Transfection Trypanosoma cruzi Tubulin Protozoa Trypanosoma Trypanosoma cruzi DaRocha, W.D. Silva, R.A. Bartholomeu, D.C. Pires, S.F. Freitas, J.M. Macedo, A.M. Vazquez, M.P. Levin, M.J. Teixeira, S.M.R. Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
topic_facet |
alpha tubulin beta tubulin DNA fragment immunoglobulin luciferase red fluorescent protein ribosome protein 5' untranslated region article electroporation epimastigote expression vector gene expression microbial genetics nonhuman priority journal reporter gene Trypanosoma cruzi 5' Untranslated Regions Animals Base Sequence Electroporation Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luciferases Luminescent Proteins Molecular Sequence Data Protozoan Proteins Transfection Trypanosoma cruzi Tubulin Protozoa Trypanosoma Trypanosoma cruzi |
description |
To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5′ untranslated plus intergenic (5′ UTR plus Ig) regions from GAPDH, TcP2β, α- and β-tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5′ UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite. |
format |
JOUR |
author |
DaRocha, W.D. Silva, R.A. Bartholomeu, D.C. Pires, S.F. Freitas, J.M. Macedo, A.M. Vazquez, M.P. Levin, M.J. Teixeira, S.M.R. |
author_facet |
DaRocha, W.D. Silva, R.A. Bartholomeu, D.C. Pires, S.F. Freitas, J.M. Macedo, A.M. Vazquez, M.P. Levin, M.J. Teixeira, S.M.R. |
author_sort |
DaRocha, W.D. |
title |
Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
title_short |
Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
title_full |
Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
title_fullStr |
Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
title_full_unstemmed |
Expression of exogenous genes in Trypanosoma cruzi: Improving vectors and electroporation protocols |
title_sort |
expression of exogenous genes in trypanosoma cruzi: improving vectors and electroporation protocols |
url |
http://hdl.handle.net/20.500.12110/paper_09320113_v92_n2_p113_DaRocha |
work_keys_str_mv |
AT darochawd expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT silvara expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT bartholomeudc expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT piressf expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT freitasjm expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT macedoam expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT vazquezmp expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT levinmj expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols AT teixeirasmr expressionofexogenousgenesintrypanosomacruziimprovingvectorsandelectroporationprotocols |
_version_ |
1782028009850535936 |