Abstract
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.
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Acknowledgements
The authors are grateful to Dr. John E. Donelson from the University of Iowa, Iowa, USA for providing a training opportunity for WDR and valuable suggestions, Etel R. Vieira for FACS analyses, and to Alice Machado-Silva for critical reading of the manuscript. We are also thankful to Dr. Egler Chiari for providing some of the T. cruzi strains. This work was supported by funds from the World Health Organization/Special Program for Research and Training in Tropical Diseases (WHO/TDR) and the NIH Fogarty International Research Collaborative Award (FIRCA). The work of WDR, RAS, DCB, SFP and SMRT received further support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The work of MV and MJL was supported by grants from FONCYT-PICTs 01-05225 and 01-06803 TWAS research grants 00–311 RG/BIO/LA. In addition, MJL was supported by an International Research Scholar grant from the Howard Hughes Medical Institute, Chevy Chase, Md., USA. The experiments described here were performed in Brazil and comply with the current laws of the country.
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DaRocha, W.D., Silva, R.A., Bartholomeu, D.C. et al. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol Res 92, 113–120 (2004). https://doi.org/10.1007/s00436-003-1004-5
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DOI: https://doi.org/10.1007/s00436-003-1004-5