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 (2019). Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus-Host Interactions. Viruses, 11(8) Molecular Diversity Preservation International MDPI 10.3390/v11080747

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Research Foci > Host-Pathogen Interaction
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Virology and Immunology
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP)

UniBE Contributor:

Jonsdottir, Hulda Run; Marti, Sabrina; Thiel, Volker Earl and Dijkman, Ronald

Subjects:

600 Technology > 630 Agriculture
500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1999-4915

Publisher:

Molecular Diversity Preservation International MDPI

Language:

English

Submitter:

Achim Braun Parham

Date Deposited:

08 Oct 2019 14:48

Last Modified:

23 Oct 2019 09:01

Publisher DOI:

10.3390/v11080747

PubMed ID:

31412613

Uncontrolled Keywords:

human airway epithelial cell cultures respiratory viruses virus-host interactions

BORIS DOI:

10.7892/boris.133781

URI:

https://boris.unibe.ch/id/eprint/133781

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