1. Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells
- Author
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Chênais, Nathalie, Lorca, Thierry, Morin, Nathalie, Guillet, Brigitte, Rime, Hélène, Le Bail, Pierre-Yves, Labbé, Catherine, Laboratoire de Physiologie et Génomique des Poissons (LPGP), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Recherche Agronomique (INRA), UMR 5237 Centre de Recherche en Biologie Cellulaire, Centre National de la Recherche Scientifique (CNRS), UMS3387 Centre de Ressources Biologiques Xénopes, Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES), Institut National de la Recherche Agronomique (INRA)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), This work benefited from the financial support of the program 'Investissements d’Avenir' ANR-11-INBS-0003 (CRB-Anim 2013–2019), ANR-11-INBS-0003,CRB-Anim,Réseau de Centres de Ressources Biologiques pour les animaux domestiques(2011), Centre de recherche en Biologie cellulaire de Montpellier (CRBM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Rennes (UR), and Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)
- Subjects
oeuf de xenopus ,nuclear transfer ,reprogrammation nucléaire ,[SDV]Life Sciences [q-bio] ,Cell Culture Techniques ,lcsh:Medicine ,Complex Mixtures ,Article ,Xenopus laevis ,poisson ,Goldfish ,culture de cellules ,cyprinidae ,Animals ,reprogramming of the genome ,lcsh:Science ,Cells, Cultured ,foetal development ,Ovum ,fish ,amphibians ,amphibien ,lcsh:R ,Cellular Reprogramming ,cryoconservation ,xenopus ,poisson rouge ,transfert nucléaire ,cellule somatique ,xenope ,développement embryonnaire ,cryofixation ,embryonic structures ,somatic cell ,lcsh:Q - Abstract
Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.
- Published
- 2019
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