ArticleBMC microbiology2010
A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.
Article in BMC microbiology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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Who cites it
24 citing papers in PubMed.
- Trypanosoma cruzi eIF4E3- and eIF4E4-containing complexes bind different mRNAs and may sequester inactive mRNAs during nutritional stress.Nucleic acids research · 2025Article
- TcZC3HTTP, a regulatory element that contributes toMicrobiology spectrum · 2024Article
- A unique mRNA decapping complex in trypanosomes.Nucleic acids research · 2023Article
- Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes.Molecular & cellular proteomics : MCP · 2022Article
- Characterization of the RNA-Binding Protein TcSgn1 inMicroorganisms · 2021Article
- Characterising ISWI chromatin remodeler in Trypanosoma cruzi.Memorias do Instituto Oswaldo Cruz · 2020Article
- Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.BMC microbiology · 2019Article
- Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes.Memorias do Instituto Oswaldo Cruz · 2018Article
- Assessing the partners of the RBP9-mRNP complex in Trypanosoma cruzi using shotgun proteomics and RNA-seq.RNA biology · 2018Article
- Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery.Memorias do Instituto Oswaldo Cruz · 2018Article
- Article
- Conservation and divergence within the clathrin interactome of Trypanosoma cruzi.Scientific reports · 2016Article
- Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi.BMC microbiology · 2015Article
- pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation.Memorias do Instituto Oswaldo Cruz · 2015Article
- Article
- Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.Memorias do Instituto Oswaldo Cruz · 2014Article
- An orthology-based analysis of pathogenic protozoa impacting global health: an improved comparative genomics approach with prokaryotes and model eukaryote orthologs.Omics : a journal of integrative biology · 2014Article
- Clathrin expression in Trypanosoma cruzi.BMC cell biology · 2014Article
- Trypanosoma cruzi bromodomain factor 3 binds acetylated α-tubulin and concentrates in the flagellum during metacyclogenesis.Eukaryotic cell · 2014Article
- Article
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe 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 high-throughput 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.
resultsWe 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.
conclusionsWe 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.
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