SARS-CoV-2 Spike "donor" cells for cell fusion assays

HEK293-derived "donor" cell line for Spike-dependent cell fusion

InvivoGen offers a human embryonic kidney (HEK)293-derived cell line, specifically designed to study SARS-CoV-2-host cell fusion:

• 293-hMyD88 Cells

These cells have been engineered to express a  constitutively active human MyD88 [1]. Upon transient transfection, with a Spike expression plasmid, 293‑hMyD88 cells become a SARS-CoV-2 mimic, which can then fuse with InvivoGen's permissive reporter cells (e.g. HEK‑Blue™ hACE2(‑TMPRSS2) and A549‑Dual™ hACE2‑TMPRSS2) [2]. 

Cell fusion with 293-hMyD88 "donor" cells

More details

Rationale

SARS-CoV-2 variants are characterized by several mutations within the antigenic Spike protein, which drives the virus entry into target cells [3]. These mutations provide a selective advantage to the virus, ultimately reducing the effectiveness of vaccination and therapeutic strategies [4]. Thus, Spike-mediated SARS-CoV-2 fusion with target cells is a relevant readout to screen for novel neutralizing monoclonal antibodies (mAbs) or small molecule inhibitors.

InvivoGen has developed a simple cell fusion assay that relies on the transfer of the adaptor molecule, MyD88, from a 'donor cell line' to an 'acceptor cell line' expressing an NF-κB-SEAP-inducible reporter gene. Cell fusion is readily assessable in the co-culture supernatant using the SEAP detection reagent, QUANTI-Blue™ Solution.

Learn more about InvivoGen's cell fusion assays
 

Key Features

• Stable and constitutive activation of the human MyD88 gene for NF-κB-SEAP-based cell fusion assays.
• Functionally validated in cell fusion assays using pUNO1-Spike (Wuhan-Hu-1 D614) and the HEK-Blue™ hACE2(-TMPRSS2) reporter cell line.
   

Applications

• Screening inhibitors of Spike-binding and/or SARS-CoV-2 host receptors.
• Studying the efficacy of vaccination and current therapeutics against emerging SARS-CoV-2 variants.

➔ 293-hMyD88 cells with stable expression of Spike (Wuhan, original), Spike V8 (Delta/B.1.617,2), or Spike V11 (Omicron/BA.1) have also been generated. These cells have been used to compare the efficiency of EK1C4 at inhibiting SARS-CoV2 Spike variant-mediated cellular fusion. See data

Learn more about emerging SARS-CoV-2 variants

References:

1. Deguine J. & Barton G.M. 2014. MyD88: a central player in innate immune signaling. F1000Prime Reports, 6:97, doi:10.12703/P6-97
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. Garcia-Beltran, W.F. et al. 2021. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell. doi:10.1016/j.cell.2021.03.013
4. Greaney, A.J. et al., 2021. Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escapes antibody recognition. Cell Host & Microbe. DOI: 10.1016/j.chom.2020.11.007.

Figures

Assessing cell fusion with 293-hMyD88-Spike cells and HEK-Blue™ hACE2 cells. 293-hMyD88 cells were transiently transfected with pUNO1-Spike using LyoVec™. After 24 hours, the cells were washed, and a dilution series of the ‘donor’ non-transfected or the 293-hMyD88-Spike cells were co‑cultured with either 2.0 x10⁴ HEK‑Blue™ Null1-v or HEK‑Blue™ hACE2 cells. After overnight incubation, cell fusion was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as OD630nm ± SEM.

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: Puromycin

Quality Control:

• Functionally validated in cell fusion assays using pUNO1-Spike and HEK-Blue™ hACE2 cells.
• The stability for 20 passages, following thawing, has been verified for both cell lines.
• These cells are guaranteed mycoplasma-free. 

Contents

• 3-7 x 10⁶ 293-hMyD88 cells in a cryovial or shipping flask.
• 1 ml of Puromycin (10 mg/ml). Store at 4 °C or at -20 °C.
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi. Store at -20 °C.

IMPORTANT: If cells are shipped frozen (i.e. in a cryovial) and are not frozen upon arrival, contact InvivoGen immediately.

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

Details

MyD88 and InvivoGen's cell fusion assay

A gain-of-function mutant of MyD88, the canonical adaptor and central node in inflammatory signalling pathways, has been described in the literature. This mutant (L256P) can spontaneously assemble and lead to persistent NF-κB activation. Therefore, upon cell fusion in InvivoGen’s assay, the constitutively expressed MyD88 in the donor cell line is transferred to the acceptor cell, where it is able to activate an IRAK-dependent signalling cascade. Ultimately, this leads to the expression of the NF-κB-dependent SEAP reporter. 

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