Expanding the toolbox for Trypanosoma cruzi: a parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping

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dc.contributorLab. Parasitologiapt_BR
dc.contributor.authorCosta, Fernanda Cristinapt_BR
dc.contributor.authorFrancisco, Amanda Fortespt_BR
dc.contributor.authorJayawardhana, Shiromanipt_BR
dc.contributor.authorCalderano, Simone Guedespt_BR
dc.contributor.authorLewis, Michael D.pt_BR
dc.contributor.authorOlmo, Franciscopt_BR
dc.contributor.authorBeneke, Tompt_BR
dc.contributor.authorGluenz, Evapt_BR
dc.contributor.authorSunter, Jackpt_BR
dc.contributor.authorDean, Samuelpt_BR
dc.contributor.authorKelly, John Morrisonpt_BR
dc.contributor.authorTaylor, Martin Craigpt_BR
dc.date.accessioned2020-07-09T21:20:13Z-
dc.date.available2020-07-09T21:20:13Z-
dc.date.issued2018pt_BR
dc.identifier.citationCosta FC, Francisco AF, Jayawardhana S, Calderano SG, Lewis MD., Olmo F, et al. Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. PLoS Negl Trop Dis. 2018 Apr;12(4):e0006388. doi:10.1371/journal.pntd.0006388.pt_BR
dc.identifier.urihttps://repositorio.butantan.gov.br/handle/butantan/2478-
dc.description.abstractBackground Infection with Trypanosome cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite. Methodology/Principal findings Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed. Conclusions/Significance The techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a community resource and are freely available on request.pt_BR
dc.description.sponsorship(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulopt_BR
dc.description.sponsorshipBritish Heart Foundationpt_BR
dc.description.sponsorship(EU) European Unionpt_BR
dc.description.sponsorship(MRC) Medical Research Councilpt_BR
dc.description.sponsorshipWellcome Trustpt_BR
dc.format.extente0006388pt_BR
dc.language.isoEnglishpt_BR
dc.relation.ispartofPlos Neglected Tropical Diseasespt_BR
dc.rightsOpen accesspt_BR
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/pt_BR
dc.titleExpanding the toolbox for Trypanosoma cruzi: a parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotypingpt_BR
dc.typeArticlept_BR
dc.rights.licenseCC BYpt_BR
dc.identifier.doi10.1371/journal.pntd.0006388pt_BR
dc.identifier.urlhttp://dx.doi.org/10.1371/journal.pntd.0006388pt_BR
dc.contributor.externalThe London School of Hygiene & Tropical Medicine (LSHTM)¦¦Reino Unidopt_BR
dc.contributor.external(USP) Universidade de São Paulopt_BR
dc.contributor.externalUniversity of Oxford¦¦Inglaterrapt_BR
dc.contributor.externalOxford Brookes University¦¦Reino Unidopt_BR
dc.identifier.citationvolume12pt_BR
dc.identifier.citationissue4pt_BR
dc.relation.ispartofabbreviatedPLoS Negl Trop Dispt_BR
dc.identifier.citationabntv. 12, n. 4, e0006388, abr. 2018pt_BR
dc.identifier.citationvancouver2018 Apr;12(4):e0006388pt_BR
dc.contributor.butantanCalderano, Simone Guedes|:Pesquisador|:Lab. Parasitologia|:pt_BR
dc.sponsorship.butantanBritish Heart Foundation¦¦PG/13/88/30556pt_BR
dc.sponsorship.butantan(EU) European Union¦¦625810pt_BR
dc.sponsorship.butantan(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulo¦¦2013/07600-3pt_BR
dc.sponsorship.butantan(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulo¦¦2016/08958-7pt_BR
dc.sponsorship.butantan(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulo¦¦2016/21283-9pt_BR
dc.sponsorship.butantanMedical Research Council (MRC)l¦¦15/16_MSD_836338pt_BR
dc.sponsorship.butantanMedical Research Council (MRC)¦¦MR/N017323/1pt_BR
dc.sponsorship.butantanWellcome Trust¦¦108445/Z/15/Zpt_BR
dc.sponsorship.butantanWellcome Trust¦¦104627/Z/14/Zpt_BR
dc.identifier.bvsccBR78.1pt_BR
dc.identifier.bvsdbIBProdpt_BR
dc.description.dbindexedYespt_BR
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