3R-Project 128-11

Genetic modification of the human airway epithelium – a paradigmatic system to study host responses to human respiratory viruses

Ronald Dijkman and Volker Thiel
Institute of Immunobiology, Kantonal Hospital St. Gallen, 9007 St. Gallen,Switzerland
ronald.dijkman@kssg.ch, volker.thiel@kssg.ch

Keywords: human; viruses; epithelia; epithelia; lung; lung diseases; cell cultures: organ-specific; cell cultures: transgenic; reduction; replacement; infectiosity

Duration: 2 years Project Completion: 2013

Background and Aim
The human airway epithelium represents the entry port of many human respiratory viruses, such as respiratory syncytial virus, rhinoviruses, influenza viruses, human parainfluenza viruses, adenoviruses, human metapneumoviruses, and human coronaviruses. Recent advances in the cultivation of primary human airway epithelia (HAE) have enabled studies of human respiratory virus infections in a culture system that morphologically and functionally resembles human airways in vivo. However, despite these recent technical achievements, the analysis of basic virus-host interactions on the molecular level is still preferentially done in animal models. This is particularly true for virus infections for which a murine model is available. The main reason for this preference is that a murine model of viral infection allows for the use of widely available transgenic or knock-out mouse strains. Thus, in contrast to primary human cells, individual genes can be genetically deleted or trans-complemented in the mouse model, which permits detailed analyses of virus-host interactions on the molecular level. In order to overcome this limitation, we aim to render the HAE culture system amenable to genetic modifications.

Method and Results
in progress (present status)
In order to render the HAE culture system amenable to genetic modification, we will use lentiviral vectors for transgene expression and to knock down the expression of specific genes.

Figure 1: Schematic representation of HAE culture generation and genetic manipulation using lentiviral vectors.

Therefore, the research objectives of the proposed project include the following specific aims:

Generation of a set of lentiviral vectors encoding reporter proteins that are expressed upon induction.

Optimization of transduction of primary HAE cultures and establishment of efficacious affinity selection methods to obtain HAE cultures with high lentivirus vector-based gene expression.

Figure 2: Human Coronavirus-infected HAE culture. Transparent 3D-rendered Z-stack image of an HCoV-229E-infected HAE cell culture is shown. The culture was stained with antibodies directed against Beta-tubulin IV (ciliated cells; red), anti-ZO1 (tight junctions; yellow) and with DAPI (cell nucleus; blue). HCoV-229E target cells express HCoV-encoded green fluorescent protein (green). The Z-stack was acquired with an EC Plan-Neofluar 40x/1.30 Oil DIC M27 objective on a Zeiss LSM 710 confocal microscope, and data were processed using Imaris (Bitplane Scientific Software).

Conclusions and Relevance for 3R
The possibility to efficiently inactivate the expression of particular genes and to express genes of interest in trans in an human airway epithelia culture system has the potential to replace many animal experiments that are based on the use of specific transgenic and knock-out mouse strains. Therefore, we expect that an HAE culture system amenable to the genetic modification of host gene expression will find wide application in the analysis of host-pathogen interactions and the molecular biology of many human respiratory pathogens.

More indormation about most recent work you will find in the Journal mBiosphere

References
1. Dvorak, A., A. E. Tilley, R. Shaykhiev, R. Wang, and R. G. Crystal. 2011. Do airway epithelium air-liquid cultures represent the in vivo airway epithelium transcriptome? Am J Respir Cell Mol Biol 44:465-473.

2. Eriksson, K. K., D. Makia, and V. Thiel. 2008. Generation of recombinant coronaviruses using vaccinia virus as the cloning vector and stable cell lines containing coronaviral replicon RNAs. Methods Mol Biol 454:237-254.

3. Thiel, V., J. Herold, B. Schelle, and S. G. Siddell. 2001. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J Gen Virol 82:1273-1281.

4. Kindler, E., Jónsdóttir, H., Muth, D., Hamming, O., Hartmann, R., Rodriguez, R., Geffers, R., Fouchier, R.A.M., Drosten, C., Müller, M.A., Dijkman, R., and Thiel, V. 2013. Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential. mBio. doi:10.1128/mBio.00611-12