The Replication of a Mouse Adapted SARS-CoV in a Mouse Cell Line Stably Expressing the Murine SARS-CoV Receptor mACE2 Efficiently Induces the Expression of Proinflammatory Cytokines

Authors:
Jose A Regla-Nava  1 , Jose M Jimenez-Guardeño, Jose L Nieto-Torres, Thomas M Gallagher, Luis Enjuanes, Marta L DeDiego
In:
Source:
Publication Date: (2013)
Issue: :
Research Area:
Respiratory Research
Platform:
4D-Nucleofector® X-Unit
Experiment

Generation of DBT cells stably expressing mACE2: To transfect DBT-mACE2 with high efficiency, 3 ×105 DBTmACE2 cells were transfected using the program EN-158 of Amaxa 4D-Nucleofector and 1 ug of the linearized plasmid pcDNA3.1- myc-mACE2 in buffer SG (Lonza). Nucleofected cells were seeded on 24-well plates. After nucleofection, cells were incubated for 10 min at room temperature (RT), and then the cells were added to DMEM supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS, Biowhittaker / Lonza) and 1% non-essential amino acids and incubated at 37 °C. 24 h post-nucleofection, G418 was added to the culture medium, to a final concentration of 800 ug/ml, to select for geneticin resistance. The selective medium was changed every 3–4 days for 2 weeks, and the cells were cloned three times by limiting dilution (1 cell/well) in the presence of G418 (800 µg/ml). Subsequently, ten clones of DBT-mACE2 were amplified in the presence of 800 µg/ml of G418.

Abstract

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-ß, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis.