CMV-driven expression plasmid for S that was also FLAG-tagged at the COOH terminus (pcDNA-SARS-CoV-2-S) was a kind gift of Craig Wilen (Yale). Plasmid encoding human ACE2 (hACE2) was obtained from Addgene (hACE2; catalog #1786). The hACE2 2.6 kbp ORF was blunt-subcloned into pShuttle (Clontech) to make pShuttle-hACE2. It was also blunt-cloned into a third generation HIV vector 3’ of CMV promoter and 5’ of an IRES-puror cassette to generate pHIV-CMV-hACE2-IRES-Puro. A separate third generation HIV vector pLV-EF1a-hACE2-cMYC-FLAG-IRES-Puro was obtained from Craig Wilen. pSV-Tat, pCMV-Tat, and pLTR-LUC were kind gifts of Andrew Rice (Baylor College of Medicine). Spike from pcDNA-SARS-CoV-2-S was inserted into a piggybac transposon (originally obtained from Matt Wilson of Baylor, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; resultant plasmid was named pT-PB-SARS-CoV-2-Spike-IRES-Blasti.
Hace2
Manufactured by Addgene
Sourced in Italy
HACE2 is a laboratory equipment product. It is a histone acetyltransferase enzyme that catalyzes the acetylation of histones, which are proteins that play a role in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Tmprss2, by Addgene (2 mentions)
Pshuttle, by Takara Bio (1 mentions)
Gblock dna fragment, by Integrated DNA Technologies (1 mentions)
Plenti dsred ires egfp, by Addgene (1 mentions)
Pcmv vsv g, by Addgene (1 mentions)
Pcmv dr8.2 dvpr, by Addgene (1 mentions)
Plenti cmv hygro dest, by Addgene (1 mentions)
Ecorv cut plenti cmv puro dest, by Addgene (1 mentions)
Padvantage, by Promega (1 mentions)
Nebuilder hifi dna assembly master mix, by Addgene (1 mentions)
Fugene hd, by Promega (1 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
Gfp1 10 plasmid, by Addgene (1 mentions)
Gfp11, by Addgene (1 mentions)
Bx73 fluorescence microscope, by Olympus (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi h1200, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
8 protocols using hace2
1
Lentiviral and Transposon Expression of SARS-CoV-2 Spike Protein
2
Construction of SARS-CoV-2 Spike Expression Plasmid
3
Generating SARS-CoV-2 Spike Protein Expressing Cell Lines
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Plasmid encoding human ACE2 (hACE2) was obtained from Addgene (hACE2; catalog #1786). The hACE2 2.6 kbp ORF was also blunt-cloned into a third generation HIV vector 3’ of CMV promoter and 5’ of an IRES-puror cassette to generate pHIV-CMV-hACE2-IRES-Puro. It was inserted into a piggybac transposon (Matt Wilson of Baylor College of Medicine, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; resultant plasmid was named pT-PB-SARS-CoV-2-Spike-IRES-Blasti. This too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES-Blasti.
4
Optimizing Viral Fusion Assays
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rLuc-GFP 1–7 and rLuc-GFP 8–11 plasmids (available upon request under MTA from Zene Matsuda, University of Tokyo [20 (link)]) were used to test the stable cell lines that were generated. RSV-specific fusion assay optimization and mFITs in combination with mAbs were carried out using bRSV-F (Snook strain, Y17970.1) or a codon-optimized hRSV-F (A2 strain, EF566942.1). pGEN2.1 plasmids expressing codon-optimized NiV-F and NiV-G ORFs (Malaysia strain, AY816748.1 and AY816745.1, respectively), tagged at their C termini with haemagglutinin (HA) and myc, respectively, were used for initial optimization of fusion assays and subsequently used in an equivalent ratio for mFIT and mVNT assays in combination with animal sera or mAbs. pcDNA3.1 plasmids expressing codon-optimized SARS-CoV-2 spike, S (Wuhan strain QHR63290.2) or SARS-CoV S (ShanghaiQXC2 strain, AAR86775.1), tagged at their C termini with FLAG, and human angiotensin-converting enzyme 2 (hACE2, Addgene), were used to optimize fusion assays and subsequently used in mFITs and mVNTs with mAbs or human plasma.
5
Lentivirus Production for SARS-CoV-2 Proteins
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The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe), TMPRSS2 (Addgene, #53887, gift from Roger Reeves), TMEM106B (Genscript, OHu17671) and VAC14 (Addgene, #47418, gift from Peter McPherson).
Individual cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST Addgene, #17452, gift from Eric Campeau & Paul Kaufman) or plenti-CMV-Hygro-DEST (Addgene, #17454, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.
Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48h post-transfection, filtered and added to recipient cells in presence of Polybrene (SCBT). Transduced cells were subsequently selected using Puromycin or Hygromycin for 5–7 days.
Individual cDNAs were cloned into EcoRV-cut plenti-CMV-Puro-DEST Addgene, #17452, gift from Eric Campeau & Paul Kaufman) or plenti-CMV-Hygro-DEST (Addgene, #17454, gift from Eric Campeau & Paul Kaufman) using NEBuilder HiFi DNA Assembly Master Mix (NEB). To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.
Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega). Supernatants were collected 48h post-transfection, filtered and added to recipient cells in presence of Polybrene (SCBT). Transduced cells were subsequently selected using Puromycin or Hygromycin for 5–7 days.
6
Generating Cell Lines Expressing Human ACE2 and SARS-CoV-2 Spike
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Plasmid encoding human ACE2 (hACE2) was obtained from Addgene (hACE2; catalog #1786). The hACE2 2.6 kbp ORF was also blunt-cloned into a third-generation HIV vector 3′ of the CMV promoter and 5′ of an IRES-puror cassette to generate pHIV-CMV-hACE2-IRES-Puro. It was inserted into a piggybac transposon (Matt Wilson of Baylor College of Medicine, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; the resultant plasmid was named pT-PB-SARS-CoV-2 Spike-IRES-Blasti. This too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES-Blasti.
7
Generation of ACE2 and SARS-CoV-2 S Expressing Cells
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The expression plasmid pMD2ACE2iPuror containing the human angiotensin-converting enzyme (ACE2) cDNA used to generate ACE2 positive cells was constructed as follows: the ACE2 PmeI cDNA fragment obtained from the plasmid hACE2 (Addgene; #1786) was cloned in pMD2iPuror opened in EcoRV.
The SARS-CoV-2 S gene from the Wuhan-Hu-1 isolate (GenBank: MN908947.3) was codon optimized (Genscript, Township, NJ) and cloned in pMD2iPuror in EcoRI/XhoI. A shorter version with a 19-codon deletion in C-terminal (ΔS) was also constructed in a similar way. These sequences will be made available upon request.
The pMD2GPiZeor, pMD2.GalviPuror and pMD2.G plasmids that encode MLV Gag-pol, and the Galv and VSV-G envelopes have been described elsewhere (Ghani et al., 2007 (link)). RetroVec is a retroviral vector plasmid containing the GFP gene under the control of the 5′ long terminal repeat sequence and that was derived from GFP3 (Qiao et al., 2002 (link)).
The SARS-CoV-2 S gene from the Wuhan-Hu-1 isolate (GenBank: MN908947.3) was codon optimized (Genscript, Township, NJ) and cloned in pMD2iPuror in EcoRI/XhoI. A shorter version with a 19-codon deletion in C-terminal (ΔS) was also constructed in a similar way. These sequences will be made available upon request.
The pMD2GPiZeor, pMD2.GalviPuror and pMD2.G plasmids that encode MLV Gag-pol, and the Galv and VSV-G envelopes have been described elsewhere (Ghani et al., 2007 (link)). RetroVec is a retroviral vector plasmid containing the GFP gene under the control of the 5′ long terminal repeat sequence and that was derived from GFP3 (Qiao et al., 2002 (link)).
8
SARS-CoV-2 Spike Protein Cell Fusion Assay
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293T cells were co-transfected with different SARS-CoV-2 spike plasmids with GFP1-10 plasmid (cat#68715, Addgene) as effector cells. Another population of 293T cells was co-transfected with human ACE2 (hACE2), TMPRSS2, and GFP11 (cat#68716, Addgene) as target cells. After 24 h post-transfection, the effector and target cells were digested by EDTA-Trypsin (25200072, Gibco) and mixed at a 1:1 ratio. The mixed cells were co-cultured at a 37°C incubator for another 24 h. The mixed cells were fixed in 10% formalin and then permeabilized with 0.1% Triton-X100 (11332481001, Sigma, USA) at room temperature. The antifade mounting medium with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, H-1200, Vector Laboratories) was used for mounting and DAPI staining. Images were taken with the Olympus BX73 fluorescence microscope (Olympus Life Science, Tokyo, Japan). The fusion area of images was quantified by ImageJ.
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