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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Bibliographic Details
Published in:Parasitology research (1987) 2004-01, Vol.92 (2), p.113-120
Main Authors: 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
Format: Article
Language:English
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
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Summary: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.
ISSN:0932-0113
1432-1955
DOI:10.1007/s00436-003-1004-5