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A549-Dual™ KO-MDA5 Cells for SARS-CoV-2 studies

A549-Dual™ KO-MDA5 hACE2-TMPRSS2 Cells Unit size Cat. code Docs Qty Price
MDA5 knockout - Reporter Human Lung Carcinoma
3-7 x 10e6 cells
a549d-komda5-cov2r
+-
$1,667.00
A549-Dual™ hACE2-TMPRSS2 Cells Unit size Cat. code Docs Qty Price
Reporter Human Lung Carcinoma
3-7 x 10e6 cells
a549d-cov2r
+-
$1,515.00

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

Reporter systems in A549-Dual™-derived cells
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

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.

More details


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) endogenously or are knockout for this gene. MDA5 is a sensor of viral RNA that has been reported to participate in the immune response to SARS-CoV-2 infection [1, 2].


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, 4]. 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 [3-5, 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

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 on SARS-CoV-2Learn 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. 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.
4. 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.
5. Matsuyama S. et al., 2020. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. PNAS. 117(13):7001-7003.

Figures

Validation of ACE2 and TMPRSS2 overexpression by RT-qPCR
Validation of ACE2 and TMPRSS2 overexpression by RT-qPCR

Human ACE2 and TMPRSS2 mRNA expression in A549-Dual™ hACE2-TMPRSS2 cells.
Total mRNA was extracted from ~1x106 A549-Dual™ (parental) and A549-Dual™ hACE2-TMPRSS2 cells. ACE2 and TMPRSS2 mRNA were amplified using quantitative RT-qPCR. Data are represented as the log2 fold change comparing ACE2 or TMPRSS2 expression to a housekeeping gene.

Validation of ACE2 surface expression by FACS
Validation of ACE2 surface expression by FACS

Surface expression of hACE2 by A549-Dual™ hACE2-TMPRSS2 cells.
~2x105 A549-Dual™ (parental) and A549-Dual™ hACE2-TMPRSS2 cells were incubated with 1 µg of Spike-S1-Fc or CTLA-4-Fc fusion proteins for 1h at 4°C. Cells were then washed and incubated with 0.5 µg of a goat anti-hIgG1-Fc antibody coupled to PE for 1h at 4°C. Cell surface staining was analyzed by flow cytometry.

Infection of A549-Dual™ hACE2-TMPRSS2 cells by SARS-CoV-2 Spike pseudotyped lentiviral particles
Infection of A549-Dual™ hACE2-TMPRSS2 cells by SARS-CoV-2 Spike pseudotyped lentiviral particles

Infection of A549-Dual™ hACE2-TMPRSS2 cells by Spike (D614 or G614) pseudotyped lentiviral particles.
~1x104 (A) A549-DualT™ (parental) and (B) A549-Dual™ hACE2-TMPRSS2 cells were cultured in the presence of SARS-CoV-2 Spike D614-variant (up panels) or Spike G614-variant (bottom panels) pseudotyped GFP lentiviral particles. After 72h, the transduction efficiency of the Spike pseudotyped GFP particles was evaluated by fluorescence microscopy. Syncytia are indicated with white arrows.

Validation of ACE2 and TMPRSS2 overexpression by RT-qPCR
Validation of ACE2 and TMPRSS2 overexpression by RT-qPCR

Human ACE2 and TMPRSS2 mRNA expression in A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells.
Total mRNA was extracted from ~1x106 A549-Dual™ hACE2-TMPRSS2 (parental) and A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells. ACE2 and TMPRSS2 mRNA were amplified using quantitative RT-qPCR. Data are represented as the log2 fold change comparing ACE2 or TMPRSS2 expression to a housekeeping gene.

Validation of ACE2 surface expression by FACS
Validation of ACE2 surface expression by FACS

 Surface expression of hACE2 by A549-Dual™ KO-MDA5 -hACE2-TMPRSS2 cells.
~2x105 A549-Dual™ KO-MDA5 and A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells were incubated with 1 µg of Spike-S1-Fc or CTLA-4-Fc fusion proteins for 1h at 4°C. Cells were then washed and incubated with 0.5 µg of a goat anti-hIgG1-Fc antibody coupled to PE for 1h at 4°C. Cell surface staining was analyzed by flow cytometry.

Validation of MDA5 knockout by PCR
Validation of MDA5 knockout by PCR

Validation of MDA5 KO in A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells.
The targeted MDA5 gene in A549-Dual™ (WT), A549-Dual™ KO-MDA5, and A549-Dual™ KO-MDA5-ACE2-TMPRSS2 cells was amplified by PCR.
A549-Dual™ KO-MDA5 and A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells feature a biallelic deletion (arrow).

Infection of A549-Dual™-derived cells by SARS-CoV-2 Spike (G614) pseudotyped lentiviral particles
Infection of A549-Dual™-derived cells by SARS-CoV-2 Spike (G614) pseudotyped lentiviral particles

Infection of A549-Dual™ -derived cells by Spike (G614) pseudotyped lentiviral particles.
~1x104  (A) A549-Dual™ , (B) A549-Dual™ hACE2-TMPRSS2, (C) A549-Dual™ KO-MDA5, and (D) A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells were cultured in the presence of SARS-CoV-2 Spike G614-variant pseudotyped GFP lentiviral particles. After 72h, the transduction efficiency of the Spike pseudotyped GFP particles was evaluated by fluorescence microscopy. Syncytia are indicated with white arrows.

 Functional validation of IRF and NF-κB reporter systems in A549-Dual™ KO-MDA5-ACE2-TMPRSS2 cells
 Functional validation of IRF and NF-κB reporter systems in A549-Dual™ KO-MDA5-ACE2-TMPRSS2 cells

Infection of A549-Dual™ -derived cells by Spike (G614) pseudotyped lentiviral particles.
5x105 A549-Dual™ hACE2-TMPRSS2 or A549-Dual™ KO-MDA5-hACE2-TMPRSS2 cells were incubated with 100 ng/ml poly(dA:dT) complexed with LyoVec™, 100 ng/ml 3p-hpRNA complexed with LyoVec™, 100 ng/ml poly(I:C)HMW complexed with LyoVec™, or 10ng/ml hTNF-α. After overnight incubation, IRF responses were assessed by measuring the Lucia luciferase bioluminescent activity in the supernatant using QUANTI-Luc™. Activity normalized on response upon incubation with 103 U/ml hIFNβ is shown (A). The NF-κB activity in A549-Dual™-derived cells was assessed by measuring the SEAP activity in the supernatant using QUANTI-Blue™ Solution. Reading of optical density (OD) at 630 nm is shown (B).

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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: BlasticidinHygromycinPuromycin, 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.
  • 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).

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Contents

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

  • 3-7 x 106 A549-Dual™ hACE2-TMPRSS2 cells OR A549-Dual™ KO-MDA5-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 pouch of QUANTI-Luc™

 Shipped on dry ice (Europe, USA & Canada)

 

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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.

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