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Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi

The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of fil...

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Autores principales: Delgado-Ramos, Lidia, Marcos, Ana T., Ramos-Guelfo, María S., Sánchez-Barrionuevo, Leyre, Smet, Francis, Chávez, Sebastián, Cánovas, David
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232552/
https://www.ncbi.nlm.nih.gov/pubmed/25239104
http://dx.doi.org/10.1534/g3.114.014357
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author Delgado-Ramos, Lidia
Marcos, Ana T.
Ramos-Guelfo, María S.
Sánchez-Barrionuevo, Leyre
Smet, Francis
Chávez, Sebastián
Cánovas, David
author_facet Delgado-Ramos, Lidia
Marcos, Ana T.
Ramos-Guelfo, María S.
Sánchez-Barrionuevo, Leyre
Smet, Francis
Chávez, Sebastián
Cánovas, David
author_sort Delgado-Ramos, Lidia
collection PubMed
description The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.
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spelling pubmed-42325522014-11-18 Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi Delgado-Ramos, Lidia Marcos, Ana T. Ramos-Guelfo, María S. Sánchez-Barrionuevo, Leyre Smet, Francis Chávez, Sebastián Cánovas, David G3 (Bethesda) Investigations The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings. Genetics Society of America 2014-09-19 /pmc/articles/PMC4232552/ /pubmed/25239104 http://dx.doi.org/10.1534/g3.114.014357 Text en Copyright © 2014 Delgado-Ramos et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Delgado-Ramos, Lidia
Marcos, Ana T.
Ramos-Guelfo, María S.
Sánchez-Barrionuevo, Leyre
Smet, Francis
Chávez, Sebastián
Cánovas, David
Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title_full Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title_fullStr Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title_full_unstemmed Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title_short Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi
title_sort flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232552/
https://www.ncbi.nlm.nih.gov/pubmed/25239104
http://dx.doi.org/10.1534/g3.114.014357
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