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 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.
DaRocha
Wanderson D
WD
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, MG 31270-010, Belo Horizonte, Brazil.
Silva
Rosiane A
RA
Bartholomeu
Daniella C
DC
Pires
Simone F
SF
Freitas
Jorge M
JM
Macedo
Andrea M
AM
Vazquez
Martin P
MP
Levin
Mariano J
MJ
Teixeira
Santuza M R
SM
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D018274:Electroporation / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics; D000097573:Red Fluorescent Protein
D020121:5' Untranslated Regions; D049452:Green Fluorescent Proteins; D008156:Luciferases; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D014404:Tubulin; D000097573:Red Fluorescent Protein