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10.3389/fmicb.2021.701198

http://scihub22266oqcxt.onion/10.3389/fmicb.2021.701198
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suck abstract from ncbi


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pmid34394046      Front+Microbiol 2021 ; 12 (ä): 701198
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  • Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication #MMPMID34394046
  • Widera M; Wilhelm A; Toptan T; Raffel JM; Kowarz E; Roesmann F; Grozinger F; Siemund AL; Luciano V; Kulp M; Reis J; Bracharz S; Pallas C; Ciesek S; Marschalek R
  • Front Microbiol 2021[]; 12 (ä): 701198 PMID34394046show ga
  • 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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