Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.
Genetic Engineering of Trypanosoma (Dutonella) vivax and In Vitro Differentiation under Axenic Conditions
Simon D’Archivio,M. Médina,A. Cosson,N. Chamond,B. Rotureau,P. Minoprio,S. Goyard
Published 2011 in PLoS Neglected Tropical Diseases
ABSTRACT
PUBLICATION RECORD
- Publication year
2011
- Venue
PLoS Neglected Tropical Diseases
- Publication date
2011-12-01
- Fields of study
Biology, Medicine
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- constitutive expression
Persistent, ongoing gene expression driven by the vector without requiring stage-specific induction in the cultured parasites.
Aliases: constitutive promoter activity
- epimastigote transfection
The introduction of the expression vector into Trypanosoma vivax epimastigote cells for genetic manipulation.
Aliases: transfection of epimastigotes
- immunocompetent rodents
Rodent hosts with intact immune function used here to assess whether transfected parasites remain infectious in vivo.
Aliases: immunocompetent mice, immunocompetent rats
- luciferase reporter gene
A reporter gene inserted into the expression vector to provide a measurable expression readout in Trypanosoma vivax cells.
Aliases: luciferase
- metacyclic infective forms
The infective developmental stage of Trypanosoma vivax produced through differentiation in culture.
Aliases: metacyclic forms
- metacyclogenesis
The developmental process by which Trypanosoma vivax progresses toward its infective metacyclic stage.
Aliases: metacyclic differentiation
- stably transfected mutants
Parasite lines that have integrated or maintained the introduced construct and can be selected and propagated after transfection.
Aliases: stable transformants, transfected mutants
- trypanosoma vivax epimastigote axenic cultures
Axenic in vitro cultures of the non-infective epimastigote stage of Trypanosoma vivax used for maintenance and manipulation.
Aliases: T. vivax epimastigote axenic cultures, epimastigote axenic cultures
- t. vivax-specific expression vector
A plasmid construct designed for expression in Trypanosoma vivax and used here as the first species-specific vector for this parasite.
Aliases: Trypanosoma vivax-specific expression vector, first T. vivax specific expression vector
REFERENCES
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