Human ACE2 expressing HEK293 cells
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SEAP reporter HEK293 cells expressing human ACE2
3-7 x 10e6 cells
HEK293 NF-κB-reporter cells expressing the SARS-CoV-2 receptor ACE2
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InvivoGen offers HEK-Blue™ hACE2 cells which express high levels of the human (h)ACE2 receptor at their cell surface. ACE2 (angiotensin I-converting enzyme-2) is a type I membrane protein that is established as the host receptor for the Spike (S) protein of severe acute respiratory syndrome coronaviruses, SARS-CoV, and SARS-CoV-2 [1-5].
HEK-Blue™ hACE2 cells were generated from HEK-Blue™ Null1-v cells, which derive from the human embryonic kidney 293 (HEK-293) cell line. These cells also express an NF-κB inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene.
The addition of ACE2 alone makes HEK-Blue™ hACE2 cells permissive to infection by pseudotyped lentiviral particles expressing the SARS-CoV-2 Spike protein [6, in-house data].
Features of HEK-Blue™ hACE2 Cells:
- Verified overexpression and cell surface expression of human ACE2 by FACS using a Spike-S1-Fc fusion protein
- Functionally-tested in infection assays with pseudotyped lentiviral particles expressing the SARS-CoV-2 Spike protein
- Readily assessable NF-κB-dependent SEAP reporter activity
Applications of HEK-Blue™ hACE2 Cells:
- Screening of small molecule inhibitors and/or neutralizing antibodies of the ACE2‑Spike interaction
- Assessing NF-κB activation upon SARS-CoV or SARS-CoV-2 infection
ACE2 plays a critical role in the pathogenesis of coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 (2019-nCoV) by facilitating viral entry through its interaction with the Spike receptor-binding domain (RBD) . Following this, host proteases (e.g. transmembrane protease serine 2 (TMPRSS2)) cleave 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 [3-5]. Notably, TMPRSS2 is not needed for infection of HEK293 cells by SARS-CoV-2 spike-pseudotyped lentiviral particles [6, in-house data].
1. Hamming I. et al., 2004. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. J. Pathol. 203:631-637.
2. Li W. et al., 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 426(6965):450-454.
3. 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.
4. Zhou P. et al., 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 579(7798):270-273.
5. Walls A.C. et al., 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 181(2):281-292.e6.
6. Korber B. et al., 2020. Tracking changes in SARS-CoV-2-Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. DOI: 10.1016/j.cell.2020.06.043.
hACE2 mRNA expression in HEK-Blue™ hACE2 cells. Total mRNA was extracted from ~5x105 HEK-Blue™ Null1-v and HEK‑Blue™ hACE2 cells and ACE2 mRNA was amplified using quantitative (q)RT-PCR. Data are represented as the log2 fold change comparing hACE2 expression to a housekeeping gene.
Surface expression of hACE2 by HEK-Blue™ hACE2 cells. ~5x105 HEK-Blue™ Null1-v and HEK-Blue™ hACE2 cells were incubated with 1 μg of Spike-S1-Fc or CTLA-4-Fc fusion proteins for 1 hr at 4°C. Cells were then washed and incubated with 0.5 μg of a goat anti-hIgG1-Fc antibody coupled to PE for 1 hr at 4°C. Cell surface staining was analyzed by flow cytometry.
Specific infection of HEK-Blue™ hACE2 cells by Spike pseudotyped lentiviral particles. ~2.5x105 HEK-Blue™ Null1-v and HEK-Blue™ hACE2 cells were cultured in the presence of SARS-CoV-2 Spike (D614)-pseudotyped GFP lentiviral particles. The particles were generated using InvivoGen's pLV-SARS2-S-d19 plasmid. After 72 hr, the transduction efficiency of the Spike pseudotyped GFP particles was evaluated by fluorescence microscopy.
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™
- ACE2 gene expression has been verified by qRT-PCR, FACS staining, and functional assays.
- Activation of the NF-κB response has been verified upon stimulation with various inducers (e.g. TNF-α, IL-1β).
- The stability for 20 passages, following thawing, has been verified.
- These cells are guaranteed mycoplasma-free.
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- 3-7 x 106 HEK-Blue™ hACE2 cells in a cryovial or shipping flask
- 1 ml of Zeocin™ (100 mg/ml)
- 1 ml of Puromycin (10 mg/ml)
- 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
- 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)