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Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airw...

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Autores principales: Jonsdottir, Hulda R., Marti, Sabrina, Geerts, Dirk, Rodriguez, Regulo, Thiel, Volker, Dijkman, Ronald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723040/
https://www.ncbi.nlm.nih.gov/pubmed/31412613
http://dx.doi.org/10.3390/v11080747
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author Jonsdottir, Hulda R.
Marti, Sabrina
Geerts, Dirk
Rodriguez, Regulo
Thiel, Volker
Dijkman, Ronald
author_facet Jonsdottir, Hulda R.
Marti, Sabrina
Geerts, Dirk
Rodriguez, Regulo
Thiel, Volker
Dijkman, Ronald
author_sort Jonsdottir, Hulda R.
collection PubMed
description Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus–host interactions in human respiratory epithelium.
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spelling pubmed-67230402019-09-10 Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions Jonsdottir, Hulda R. Marti, Sabrina Geerts, Dirk Rodriguez, Regulo Thiel, Volker Dijkman, Ronald Viruses Article Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus–host interactions in human respiratory epithelium. MDPI 2019-08-13 /pmc/articles/PMC6723040/ /pubmed/31412613 http://dx.doi.org/10.3390/v11080747 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jonsdottir, Hulda R.
Marti, Sabrina
Geerts, Dirk
Rodriguez, Regulo
Thiel, Volker
Dijkman, Ronald
Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title_full Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title_fullStr Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title_full_unstemmed Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title_short Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions
title_sort establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus–host interactions
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723040/
https://www.ncbi.nlm.nih.gov/pubmed/31412613
http://dx.doi.org/10.3390/v11080747
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