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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Optimising genetic transformation of Trypanosoma cruzi using hydroxyurea-induced cell-cycle synchronisation

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Olmo, Francisco [1] ; Costa, Fernanda C. [1, 2] ; Mann, Gurdip Singh [1] ; Taylor, Martin C. [1] ; Kelly, John M. [1]
Total Authors: 5
[1] London Sch Hyg & Trop Med, Dept Pathogen Mol Biol, Keppel St, London WC1E 7HT - England
[2] Univ Sao Paulo, Inst Phys Sao Carlos, BR-13563120 Sao Carlos, SP - Brazil
Total Affiliations: 2
Document type: Journal article
Source: Molecular and Biochemical Parasitology; v. 226, p. 34-36, DEC 2018.
Web of Science Citations: 1

The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosome cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block. We found a significant increase in transformation efficiency, with both episomal and integrative constructs, when cultures were electroporated 1 h after hydroxyurea removal. It was possible to generate genetically modified populations in less than 2 weeks, compared to the normal 4-6 weeks, with a 5 to 8-fold increase in the number of stably transformed clones. This straightforward optimisation step can be widely applied and should help streamline functional studies in T. cruzi. (AU)

FAPESP's process: 13/07600-3 - CIBFar - Center for Innovation in Biodiversity and Drug Discovery
Grantee:Glaucius Oliva
Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 16/08958-7 - Exploring the use of new genome editing technology as a fast-track approach to studying topoisomerase biology in Trypanosoma cruzi
Grantee:Fernanda Cristina Costa
Support type: Scholarships abroad - Research Internship - Post-doctor