TY - JOUR
T1 - Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi
AU - Padmanabhan, Prasad K.
AU - Polidoro, Rafael B.
AU - Barteneva, Natasha S.
AU - Gazzinelli, Ricardo T.
AU - Burleigh, Barbara A.
N1 - Funding Information:
We thank members of the Burleigh lab and the Molecular Parasitology group at HSPH for helpful discussions. R.P.B. was supported by CNPq fellowship #219331/2013-8 . R.T.G. was a recipient of a Scholar Fellowship from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), Brazil , and the David Rockefeller Center for Latin American Studies at Harvard School of Public Health, USA .
Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2014/12
Y1 - 2014/12
N2 - The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field.
AB - The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field.
KW - Fluorescence-activated cell sorting
KW - Mammalian cell infection
KW - Trypanosoma cruzi
KW - Trypomastigote transfection
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U2 - 10.1016/j.molbiopara.2015.02.001
DO - 10.1016/j.molbiopara.2015.02.001
M3 - Article
C2 - 25712770
AN - SCOPUS:84924058473
VL - 198
SP - 100
EP - 103
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
SN - 0166-6851
IS - 2
ER -