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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...
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| Published in: | Parasitology research (1987) 2004-01, Vol.92 (2), p.113-120 |
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| Main Authors: | , , , , , , , , |
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| Language: | English |
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| cited_by | cdi_FETCH-LOGICAL-c327t-c82c141d610715b3e836c6b71136003a8e120a41f1a4ac3eed6bdc5d9782f6e3 |
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| container_end_page | 120 |
| container_issue | 2 |
| container_start_page | 113 |
| container_title | Parasitology research (1987) |
| container_volume | 92 |
| creator | DAROCHA, Wanderson D SILVA, Rosiane A BARTHOLOMEU, Daniella C PIRES, Simone F FREITAS, Jorge M MACEDO, Andrea M VAZQUEZ, Martin P LEVIN, Mariano J TEIXEIRA, Santuza M. R |
| 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, TcP2beta, alpha- and beta- 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. |
| doi_str_mv | 10.1007/s00436-003-1004-5 |
| format | article |
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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.</description><identifier>ISSN: 0932-0113</identifier><identifier>EISSN: 1432-1955</identifier><identifier>DOI: 10.1007/s00436-003-1004-5</identifier><identifier>PMID: 14634799</identifier><identifier>CODEN: PARREZ</identifier><language>eng</language><publisher>Berlin: Springer</publisher><subject>5' Untranslated Regions ; Animals ; Base Sequence ; Biochemistry. Physiology. Immunology. Molecular biology ; Biological and medical sciences ; Electroporation - methods ; Fundamental and applied biological sciences. Psychology ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; Luciferases - genetics ; Luciferases - metabolism ; Luminescent Proteins - genetics ; Luminescent Proteins - metabolism ; Molecular Sequence Data ; Protozoa ; Protozoan Proteins - genetics ; Protozoan Proteins - metabolism ; Red Fluorescent Protein ; Transfection ; Trypanosoma cruzi - genetics ; Trypanosoma cruzi - metabolism ; Tubulin - genetics</subject><ispartof>Parasitology research (1987), 2004-01, Vol.92 (2), p.113-120</ispartof><rights>2004 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c327t-c82c141d610715b3e836c6b71136003a8e120a41f1a4ac3eed6bdc5d9782f6e3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15476805$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14634799$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>DAROCHA, Wanderson D</creatorcontrib><creatorcontrib>SILVA, Rosiane A</creatorcontrib><creatorcontrib>BARTHOLOMEU, Daniella C</creatorcontrib><creatorcontrib>PIRES, Simone F</creatorcontrib><creatorcontrib>FREITAS, Jorge M</creatorcontrib><creatorcontrib>MACEDO, Andrea M</creatorcontrib><creatorcontrib>VAZQUEZ, Martin P</creatorcontrib><creatorcontrib>LEVIN, Mariano J</creatorcontrib><creatorcontrib>TEIXEIRA, Santuza M. R</creatorcontrib><title>Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols</title><title>Parasitology research (1987)</title><addtitle>Parasitol Res</addtitle><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, TcP2beta, alpha- and beta- 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.</description><subject>5' Untranslated Regions</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Biochemistry. Physiology. Immunology. Molecular biology</subject><subject>Biological and medical sciences</subject><subject>Electroporation - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes, Reporter</subject><subject>Genetic Vectors</subject><subject>Green Fluorescent Proteins</subject><subject>Luciferases - genetics</subject><subject>Luciferases - metabolism</subject><subject>Luminescent Proteins - genetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Protozoa</subject><subject>Protozoan Proteins - genetics</subject><subject>Protozoan Proteins - metabolism</subject><subject>Red Fluorescent Protein</subject><subject>Transfection</subject><subject>Trypanosoma cruzi - genetics</subject><subject>Trypanosoma cruzi - metabolism</subject><subject>Tubulin - genetics</subject><issn>0932-0113</issn><issn>1432-1955</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><recordid>eNpFkE9PwzAMxSMEYmPwAbigXOBWiJs0bbmhafyRJnHZvcpSdypqmxK308anJ9Mm7WQ_6edn-zF2D-IZhEhfSAgldSSEjIJWUXLBpqBkHEGeJJdsKvLQCwA5YTdEP0JAqpW6ZhNQWqo0z6fMLna9R6LaddxVHHdug50biYeCxOuOr_y-N50j1xpu_fhXv_K67b3b1t2Gb9EOzhM3XcmxCcK73nkzHOwCMzjrGrplV5VpCO9OdcZW74vV_DNafn98zd-WkZVxOkQ2iy0oKDWIFJK1xExqq9dpuF-HF02GEAujoAKjjJWIpV6XNinzNIsrjXLGno62YfHviDQUbU0Wm8Z0GD4qMgGx1CoJIBxB6x2Rx6rofd0avy9AFIdgi2OwRdh60Ko4zDyczMd1i-V54pRkAB5PgCFrmsqbztZ05hKV6kwk8h91cYJ5</recordid><startdate>20040101</startdate><enddate>20040101</enddate><creator>DAROCHA, Wanderson D</creator><creator>SILVA, Rosiane A</creator><creator>BARTHOLOMEU, Daniella C</creator><creator>PIRES, Simone F</creator><creator>FREITAS, Jorge M</creator><creator>MACEDO, Andrea M</creator><creator>VAZQUEZ, Martin P</creator><creator>LEVIN, Mariano J</creator><creator>TEIXEIRA, Santuza M. 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Molecular biology</topic><topic>Biological and medical sciences</topic><topic>Electroporation - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genes, Reporter</topic><topic>Genetic Vectors</topic><topic>Green Fluorescent Proteins</topic><topic>Luciferases - genetics</topic><topic>Luciferases - metabolism</topic><topic>Luminescent Proteins - genetics</topic><topic>Luminescent Proteins - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Protozoa</topic><topic>Protozoan Proteins - genetics</topic><topic>Protozoan Proteins - metabolism</topic><topic>Red Fluorescent Protein</topic><topic>Transfection</topic><topic>Trypanosoma cruzi - genetics</topic><topic>Trypanosoma cruzi - metabolism</topic><topic>Tubulin - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>DAROCHA, Wanderson D</creatorcontrib><creatorcontrib>SILVA, Rosiane A</creatorcontrib><creatorcontrib>BARTHOLOMEU, Daniella C</creatorcontrib><creatorcontrib>PIRES, Simone F</creatorcontrib><creatorcontrib>FREITAS, Jorge M</creatorcontrib><creatorcontrib>MACEDO, Andrea M</creatorcontrib><creatorcontrib>VAZQUEZ, Martin P</creatorcontrib><creatorcontrib>LEVIN, Mariano J</creatorcontrib><creatorcontrib>TEIXEIRA, Santuza M. 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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, TcP2beta, alpha- and beta- 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. 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| ispartof | Parasitology research (1987), 2004-01, Vol.92 (2), p.113-120 |
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| language | eng |
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| subjects | 5' Untranslated Regions Animals Base Sequence Biochemistry. Physiology. Immunology. Molecular biology Biological and medical sciences Electroporation - methods Fundamental and applied biological sciences. Psychology Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luciferases - genetics Luciferases - metabolism Luminescent Proteins - genetics Luminescent Proteins - metabolism Molecular Sequence Data Protozoa Protozoan Proteins - genetics Protozoan Proteins - metabolism Red Fluorescent Protein Transfection Trypanosoma cruzi - genetics Trypanosoma cruzi - metabolism Tubulin - genetics |
| title | Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols |
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