Visualizing SARS-CoV-2 Spike-Induced Cell Fusion

HEK293T cells were seeded into 25 cm² flasks (1 × 10⁶ cells/flask) and were transfected with pINT2-(TK-EF1α-S-ΔRS-HA-TK) or pEGFP-C1 (mock control) using polyethyleneimine (PEI) transfection reagent (Polysciences, Inc.). S-ΔRS-HA ORF was chemically synthesized by Eurofins genomics (Milano, Italy) and integrated into a pINT2 transfer vector (1 (link)) to yield pINT2-(TK-EF1α-S-ΔRS-HA-TK). pEGFP-C1 was obtained from Clontech. HEK293T cells transfection was performed as previously described (23 (link)). For syncytia formation, HEK/ACE2/TMPRRS2/Puro cells were transiently cotransfected with pINT2-(TK-EF1α-S-ΔRS-HA-TK) and pEGFP-C1 plasmids at the same molar ratio (1:1), using PEI transfection reagent as described before. Twenty-four hours after co-transfection, syncytia were observed by inverted fluorescence microscopy (Zeiss-Axiovert-S100), and images were acquired by a digital camera (Zeiss-Axiocam-MRC).

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Park S.C., Conti L., Franceschi V., Oh B., Yang M.S., Ham G., Di Lorenzo A., Bolli E., Cavallo F., Kim B, & Donofrio G. (2023). Assessment of BoHV-4-based vector vaccine intranasally administered in a hamster challenge model of lung disease. Frontiers in Immunology, 14, 1197649.

Publication 2023

PEI) transfection reagent pEGFP-C1 Axiovert-S100 Axiocam-MRC

Ace2 Camera Cells Fluorescence microscopy Molar Plasmids Polyethyleneimine S100 Syncytia Transfection Vector

Corresponding Organization : University of Parma

Other organizations : University of Turin

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Variable analysis

independent variables

Transfection of HEK293T cells with pINT2-(TK-EF1α-S-ΔRS-HA-TK) or pEGFP-C1 (mock control) using polyethyleneimine (PEI) transfection reagent

dependent variables

Syncytia formation in HEK/ACE2/TMPRRS2/Puro cells

control variables

HEK293T cell seeding density (1 × 10^6 cells/flask)
Transfection method (PEI transfection reagent)
Observation time point (24 hours after co-transfection)

controls

Positive control: pINT2-(TK-EF1α-S-ΔRS-HA-TK)
Negative control: pEGFP-C1 (mock control)

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