title: Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi.
abstract: 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.
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, United States.
authors.forename: Prasad K
authors.initials: PK
authors.lastname: Padmanabhan
authors.affiliation: Departamento de Bioquímica e Imunologia and Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Rafael B
authors.initials: RB
authors.lastname: Polidoro
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, United States.
authors.forename: Natasha S
authors.initials: NS
authors.lastname: Barteneva
authors.affiliation: Departamento de Bioquímica e Imunologia and Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Minas Gerais, Brazil; Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brazil; Division of Infectious Disease and Immunology, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, United States.
authors.forename: Ricardo T
authors.initials: RT
authors.lastname: Gazzinelli
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, United States. Electronic address: bburleig@hsph.harvard.edu.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Burleigh
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D008322:Mammals; D008967:Molecular Biology / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics*
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Fluorescence-activated cell sorting; Mammalian cell infection; Trypanosoma cruzi; Trypomastigote transfection