Expression of Exogenous Genes in Trypanosoma Cruzi: Improving Vectors and Electroporation Protocols

Overview

Journal Parasitol Res

Specialty Parasitology

Date 2003 Nov 25

PMID 14634799

Citations 44

Authors

Wanderson D DaRocha

Rosiane A Silva

Daniella C Bartholomeu

Simone F Pires

Jorge M Freitas

Andrea M Macedo

Martin P Vazquez

Mariano J Levin

Santuza M R Teixeira

Affiliations

Soon will be listed here.

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, 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.

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References

Kelly J, Ward H, Miles M, Kendall G . A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res. 1992; 20(15):3963-9. PMC: 334073. DOI: 10.1093/nar/20.15.3963. View

Vazquez M, Levin M . Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene. 1999; 239(2):217-25. DOI: 10.1016/s0378-1119(99)00386-8. View

Teixeira S, Kirchhoff L, Donelson J . Trypanosoma cruzi: suppression of tuzin gene expression by its 5'-UTR and spliced leader addition site. Exp Parasitol. 1999; 93(3):143-51. DOI: 10.1006/expr.1999.4446. View

Camargo E . GROWTH AND DIFFERENTIATION IN TRYPANOSOMA CRUZI. I. ORIGIN OF METACYCLIC TRYPANOSOMES IN LIQUID MEDIA. Rev Inst Med Trop Sao Paulo. 1964; 6:93-100. View

Coughlin B, Teixeira S, Kirchhoff L, Donelson J . Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3'-untranslated region position-dependent cis-element and an untranslated region-binding protein. J Biol Chem. 2000; 275(16):12051-60. DOI: 10.1074/jbc.275.16.12051. View

Dietrich P, Soares M, Affonso M, Floeter-Winter L . The Trypanosoma cruzi ribosomal RNA-encoding gene: analysis of promoter and upstream intergenic spacer sequences. Gene. 1993; 125(1):103-7. DOI: 10.1016/0378-1119(93)90753-p. View

Clayton C . Life without transcriptional control? From fly to man and back again. EMBO J. 2002; 21(8):1881-8. PMC: 125970. DOI: 10.1093/emboj/21.8.1881. View

Martinez-Calvillo S, Lopez I, Hernandez R . pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene. 1997; 199(1-2):71-6. DOI: 10.1016/s0378-1119(97)00348-x. View

DE Wet J, Wood K, DeLuca M, Helinski D, Subramani S . Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987; 7(2):725-37. PMC: 365129. DOI: 10.1128/mcb.7.2.725-737.1987. View

10.

LaCount D, Barrett B, Donelson J . Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis. J Biol Chem. 2002; 277(20):17580-8. DOI: 10.1074/jbc.M200873200. View

11.

Schurch N, Hehl A, Vassella E, Braun R, Roditi I . Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions. Mol Cell Biol. 1994; 14(6):3668-75. PMC: 358734. DOI: 10.1128/mcb.14.6.3668-3675.1994. View

12.

Teixeira S, Otsu K, Hill K, Kirchhoff L, Donelson J . Expression of a marker for intracellular Trypanosoma cruzi amastigotes in extracellular spheromastigotes. Mol Biochem Parasitol. 1999; 98(2):265-70. DOI: 10.1016/s0166-6851(98)00158-3. View

13.

Matthews K, Tschudi C, Ullu E . A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes. Genes Dev. 1994; 8(4):491-501. DOI: 10.1101/gad.8.4.491. View

14.

Nozaki T, Cross G . Effects of 3' untranslated and intergenic regions on gene expression in Trypanosoma cruzi. Mol Biochem Parasitol. 1995; 75(1):55-67. DOI: 10.1016/0166-6851(95)02512-x. View

15.

Vanhamme L, Pays E . Control of gene expression in trypanosomes. Microbiol Rev. 1995; 59(2):223-40. PMC: 239361. DOI: 10.1128/mr.59.2.223-240.1995. View

16.

Lebowitz J, Smith H, Rusche L, Beverley S . Coupling of poly(A) site selection and trans-splicing in Leishmania. Genes Dev. 1993; 7(6):996-1007. DOI: 10.1101/gad.7.6.996. View

17.

Weston D, La Flamme A, Van Voorhis W . Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3' untranslated, the 3' intergenic, and the coding regions. Mol Biochem Parasitol. 1999; 102(1):53-66. DOI: 10.1016/s0166-6851(99)00079-1. View

18.

Teixeira S, Russell D, Kirchhoff L, Donelson J . A differentially expressed gene family encoding "amastin," a surface protein of Trypanosoma cruzi amastigotes. J Biol Chem. 1994; 269(32):20509-16. View

19.

Cano M, Gruber A, Vazquez M, Cortes A, Levin M, Gonzalez A . Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi genome project. Mol Biochem Parasitol. 1995; 71(2):273-8. DOI: 10.1016/0166-6851(95)00066-a. View

20.

Mahmood R, Hines J, Ray D . Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata. Mol Cell Biol. 1999; 19(9):6174-82. PMC: 84551. DOI: 10.1128/MCB.19.9.6174. View