RT Journal Article
SR Electronic
T1 Establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus – host interactions
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 694380
DO 10.1101/694380
A1 Hulda R. Jonsdottir
A1 Sabrina Marti
A1 Dirk Geerts
A1 Regulo Rodriguez
A1 Volker Thiel
A1 Ronald Dijkman
YR 2019
UL http://biorxiv.org/content/early/2019/07/08/694380.abstract
AB 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 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 FACS-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As 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 (RSV) 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.