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

Autor(es)

Olmo, Francisco ^[1] ; Costa, Fernanda C. ^{[1, 2]} ; Mann, Gurdip Singh ^[1] ; Taylor, Martin C. ^[1] ; Kelly, John M. ^[1]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: Molecular and Biochemical Parasitology; v. 226, p. 34-36, DEC 2018.
• Citações Web of Science: 1

Resumo

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)

• Processo FAPESP: 13/07600-3 - CIBFar - Centro de Inovação em Biodiversidade e Fármacos
• Beneficiário: Glaucius Oliva
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 16/08958-7 - Explorando o uso de nova tecnologia de edição de genoma como uma abordagem rápida para o estudo da biologia de topoisomerases em Trypanosoma cruzi
• Beneficiário: Fernanda Cristina Costa
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado