A549-Dual™ Cells for SARS-CoV-2 studies

Dual reporter cells expressing the SARS-CoV-2 receptors ACE2 & TMPRSS2

Reporter systems in A549-Dual™-derived cells

InvivoGen offers a new series of A549-Dual™ cell lines, specifically designed for COVID-19 studies:

— A549-Dual™ hACE2-TMPRSS2 cells
— A549-Dual™ KO-MDA5 hACE2-TMPRSS2 cells
— A549-Dual™ KO-RIG-I hACE2-TMPRSS2 cells

These cells derive from the human A549 lung carcinoma cell line. They stably overexpress the genes encoding for the SARS-CoV-2 receptors, human ACE2, and TMPRSS2.

These cells also express two inducible reporter genes, allowing the concomitant study of the IRF and NF-κB pathways, by monitoring the Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase) activities, respectively. In addition, these cells either express MDA5 (Melanoma Differentiation Associated gene 5) and RIG-I (Retinoic Acid Inducible protein 1) endogenously or are knockout for each gene, individually. MDA5 and RIG-I are sensors of viral RNA that have been reported to participate in the immune response to SARS-CoV-2 infection [1-3].

The A549 cell line is commonly used for the study of respiratory infections. SARS-CoV-2, the causative agent of coronavirus disease-19 (COVID-19), gains entry into the human lung epithelium through the interaction of the virus Spike protein with the host ACE2 and TMPRSS2 receptors [3-5]. A549 cells express negligible levels of ACE2 and no TMPRSS2 and thus are poorly permissive to infection by SARS-CoV-2 or Spike pseudotyped lentiviral particles [4-6, in-house data]. To increase their permissivity, A549-Dual™ cells have been stably transfected with the human ACE2 and TMPRSS2 genes. In contrast to A549-Dual™ cells, A549-Dual™ hACE2-TMPRSS2 cells are highly permissive to Spike pseudotyped lentiviral particles as visualized by the expression of the lentiviral GFP transgene (see Figures).

Key Features:

• Overexpression  of human ACE2 and TMPRSS2 
• Highly permissive to SARS-CoV-2 Spike pseudotyped lentiviral particles
• Readily assessable Lucia luciferase and SEAP reporter activity for the IRF and NF-κB activation, respectively
• Endogenous or knockout expression of human MDA5 or human RIG-I

Applications:

• Study of SARS-CoV-2 RNA sensing pathways
• Screening of small molecule inhibitors and/or neutralizing antibodies of the ACE2‑Spike interaction
• Screening of small molecule inhibitors and/or neutralizing antibodies of the TMPRSS2 surface protease
• Comparative studies of the effects of drugs targeting ACE2 and/or TMPRSS2 on the SARS-CoV-2 infection and cellular signaling outcomes

Learn more about SARS-CoV-2 infection cycle, immune responses, and potential therapeutics.

References

1. Ying X. et al., 2021. MDA5 governs the innate immune response to SARS-CoV-2 in lung epithelial cells. Cell Reports. 34:108628.
2. Rebendenne A. et al., 2021. SARS-CoV-2 triggers MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells. J. Virol. doi:10.1128/JVI.02415-20.
3. Wu J, et al., 2021. SARS-CoV-2 ORF9b inhibits RIG-I MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Reports. 34(7):108761.
4. Chen H. et al., 2020. SARS-CoV-2 activated lung epithelia cell proinflammatory signaling and leads to immune dysregulation in COVID-19 patients by single-cell sequencing. medRxiv: DOI 10.1101/2020.05.08.20096024.
5. Hoffmann M. et al., 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 181:1-16.
6. Matsuyama S. et al., 2020. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. PNAS. 117(13):7001-7003.

Specifications

Growth medium: DMEM, 4.5 g/L glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Antibiotic resistance: Blasticidin, Hygromycin, Puromycin, and Zeocin®

Quality Control:

• ACE2 gene expression has been verified by RT-qPCR, FACS staining, and functional assays.
• TMPRSS2 gene expression has been verified by RT-qPCR, and functional assays.
• MDA5 knockout has been verified by RT-qPCR and functional assays.
• RIG-I knockout has been verified by RT-qPCR and functional assays.
• Lucia luciferase and SEAP reporter activities have been validated using functional assays.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free. 
   

InvivoGen's products are covered by a Limited Use License (See Terms and Conditions).

Contents

Please note: Each cell line is sold separately. See TDS for the exact contents of each cell line. 

• 3-7 x 10⁶A549-Dual™ hACE2-TMPRSS2 cells, OR A549-Dual™ KO-MDA5 hACE2-TMPRSS2 cells, OR A549-Dual™ KO-RIG-I hACE2-TMPRSS2 cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Puromycin (10 mg/ml)
• 1 ml of Zeocin®(100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

ACE2 AND TMPRSS2, CELL SURFACE SARS-COV-2 RECEPTORS:

ACE2 (angiotensin I-converting enzyme-2) and TMPRSS2 (transmembrane protease serine 2) play a critical role in the pathogenesis of COVID-19 by allowing viral entry into target cells (e.g. human lung epithelium). ACE2 and TMPRSS2 are cell-surface proteins that both interact with the virus Spike (S) protein [1,2-4]. ACE2 is mandatory for the binding of SARS-CoV-2 at the cell surface through its interaction with the Spike receptor-binding domain (RBD) [5]. Following this, TMPRSS2 cleaves the S protein into two functional subunits (S1 and S2), allowing virus-host membrane fusion, and the release of viral contents (e.g. RNA) into the cytosol [4-6]. Another protease, the Cathepsin L, also mediates cleavage of the S protein but it acts in the endosomes. Camostat is a clinically-proven inhibitor of TMPRSS2 and has been shown to inhibit SARS-CoV-2-S pseudotyped viral particles entry into primary human lung cells in a dose-dependent manner [2]. This observation demonstrates the critical implication of TMPRSS2 in SARS-CoV-2 infection and spread. Moreover, in lung cells that fail to express robust levels of Cathepsin L, the virus entry depends on a furin-mediated pre-cleavage of the S protein at the S1/S2 site, before subsequent TMPRSS2-mediated cleavage at the S2' site [7].

1. Chen H. et al., 2020. SARS-CoV-2 activated lung epithelia cell proinflammatory signaling and leads to immune dysregulation in COVID-19 patients by single-cell sequencing. medRxiv: DOI 10.1101/2020.05.08.20096024.
2. Hoffmann M. et al., 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 181:1-16.
3. Birra D. et al., 2020. COVID 19: a clue from innate immunity. Immunologic Research. 68(3):161-168.
4. Matsuyama S. et al., 2020. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. PNAS. 117(13):7001-7003.
5. Zhou P. et al., 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 579(7798):270-273.
6. Walls A.C. et al., 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 181(2):281-292.e6.
7. Hoffman M. et al., 2020. A multibasic cleavage site in the Spike protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular Cell. 78:1-6.

FAQ Cell Lines

Any questions about our cell lines ? Visit our frequently asked questions page