Selected article for: "acute respiratory syndrome and lung interferon"
Author: Widera, Marek; Wilhelm, Alexander; Toptan, Tuna; Raffel, Johanna M.; Kowarz, Eric; Roesmann, Fabian; Grözinger, Finn; Siemund, Anna Lena; Luciano, Vanessa; Külp, Marius; Reis, Jennifer; Bracharz, Silvia; Pallas, Christiane; Ciesek, Sandra; Marschalek, Rolf
Title: Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication
  • Cord-id: b9n3s9ti
  • Document date: 2021_7_29
    • ID: b9n3s9ti
    Snippet: 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 cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-through
    Document: 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 cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model 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 (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT 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 model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication.

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