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bioRxiv · 10.1101/2021.04.27.441606

Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

Widera, M.·Wilhelm, A.·Toptan, T.·Raffel, J. M.·Kowarz, E.·Roesmann, F.·Siemund, A. L.·Luciano, V.·Kuelp, M.·Reis, J.·Bracharz, S.·Pallas, C.·Ciesek, S.·Marschalek, R.
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Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

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Widera, M., Wilhelm, A., Toptan, T., Raffel, J. M., Kowarz, E., Roesmann, F., Siemund, A. L., Luciano, V., Kuelp, M., Reis, J., Bracharz, S., Pallas, C., Ciesek, S., Marschalek, R.. 2021-04-27. Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors. https://doi.org/10.1101/2021.04.27.441606

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