Calu-3 (cat. HTB-55), Vero E6 (cat. CRL-1586), HEK293 (cat. CRL-1573) and HEK293T (cat. CRL-3216) cells were purchased from ATCC. Calu-3 cells were grown in Eagle's minimum essential medium (EMEM) (Wisent, cat. 320–005-CL) supplemented with 15% fetal bovine serum (FBS) (Gibco, cat. 12,483–020). Vero E6 and HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS. Vero E6/TMPRSS2 cell line was generated by transducing Vero E6 cells with lentiviral particles expressing human TMPRSS2, followed by selection with 1 mg/ml G418 (Wisent, cat. 400–130-IG). HEK293/ACE2/TMPRSS2 cells were generated by sequential transduction of HEK293 cells with lentiviral particles expressing human ACE2 (selection with 2 μg/ml puromycin) and lentiviral particles expressing human TMPRSS2 (selection with 1 mg/ml G418). The TMPRSS2 (cat. 145,843) and ACE2 (cat. 145,839) lentiviral DNA clones (Rebendenne et al., 2021 ) were purchased from Addgene and transfected into HEK293T cells together with pSPAX2 (Addgene, cat. 12,260) and VSV-G DNA (Addgene, cat. 8454) (Stewart et al., 2003 (link)) to produce lentiviral particles. Virus HCoV-229E-Luc was kindly provided by Volker Thiel (van den Worm et al., 2012 (link)). Sendai virus (Cantel Strain) was obtained from Charles River Laboratories (Sze et al., 2017 ).
Vsv g dna
Manufactured by Addgene
VSV-G DNA is a plasmid containing the glycoprotein gene from the vesicular stomatitis virus (VSV-G). This gene encodes a viral envelope protein that can be used to pseudotype and package lentiviral or retroviral vectors.
Automatically generated - may contain errors
2 protocols using vsv g dna
1
Comparison of Cell Lines for SARS-CoV-2 Studies
2
Generation of HEK293-ACE2 Cell Line
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The ACE2-expressing HEK293 cell line (a gift from Dr. Chen Liang, Lady Davies Institute, McGill University, Canada) was generated by transduction with lentivirus particles that carry the human ACE2 gene. The lentivirus particles were produced by transfecting HEK293T cells with plasmid DNA pSPAX2 (encoding HIV-1 Gag-Pol, from Addgene, cat. 12260), VSV-G DNA (encoding VSV glycoprotein, from Addgene, cat. 8454) and ACE2 lentiviral DNA clone (encoding human ACE2 gene, from Addgene, cat. 145839). The transduced HEK293 cells were subject to selection with puromycin (2 µg/ml) (Thermo Fisher, J67236.XF), to select for cells that were stably transduced to express human ACE2. Cells were maintained in DMEM (Gibco; 11965-092) containing 10% FBS and 2 µg/ml puromycin. Cells were rendered quiescent by maintenance in DMEM containing 0.5% fetal bovine serum (FBS) for 2 h. Cells were split at a ratio of 1:3 and plated in 60 mm cell culture plates at a density of 0.8 × 106 cells. Our experimental protocol consisted in of stimulation of HEK293 and HEK293-ACE2 cells with rS1p (accession number: QHD43416; expressed region: Val16-Gln690; catalogue number: 230-01101-500; lot number: #09G2122L; concentration: 0.75 mg/mL; RayBiotech) for 24 h. All experiments were performed at 37 °C in 5% CO2.
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