Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/28670
Title: A high-throughput cloning system for reverse genetics in Trypanosoma cruzi
Authors: Batista, Michel
Marchini, Fabricio Klerynton
Celedon, Paola A. F.
Fragoso, Stenio Perdigão
Probst, Christian M.
Preti, Henrique
Ozaki, Luiz S.
Buck, Gregory A.
Goldenberg, Samuel
Krieger, Marco Aurélio
Affilliation: Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Center for the Study of Biological Complexity. Virginia Commonwealth University. Richmond, Virginia, USA.
Center for the Study of Biological Complexity. Virginia Commonwealth University. Richmond, Virginia, USA.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Abstract: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a highthroughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. How Resulted, we constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed. This way, we constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.
Keywords: Cloning
Genetic Vectors
Plasmids
Reverse Genetics
Keywords in spanish: Clonación Molecular
Vectores Genéticos
Plásmidos
Genética Inversa
keywords: Trypanosoma cruzi
DeCS: Clonagem Molecular
Vetores Genéticos
Plasmídeos
Genética Reversa
Issue Date: 2010
Publisher: Springer Nature
Citation: BATISTA, Michel et al. A high-throughput cloning system for reverse genetics in Trypanosoma cruzi. BMC Microbiology, v. 10, n. 259, 2010.
DOI: 10.1186/1471-2180-10-259
ISSN: 1471-2180
Copyright: open access
Appears in Collections:PR - ICC - Artigos de Periódicos

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