rAAV particles were generated by transient transfection of HEK 293T cells as previously described72 (link). In brief, 1.8 × 107 cells were plated in 15-cm dishes before transfecting the pAAV-CAG-eGFP transgene plasmid (a gift from E. Boyden, Addgene plasmid #37825), the relevant RepCap plasmid and the pAdDeltaF6 helper plasmid (a gift from J. M. Wilson, Addgene plasmid #112867), at a ratio of 10.5 µg, 10.5 µg and 30.5 µg, respectively, using PEIPro transfection reagent (PolyPlus) at a ratio of 1 µl per 1 µg DNA. Seventy-two hours post-transfection, cell pellets and supernatant were harvested and rAAV particles were purified using an Akta HPLC platform. rAAV particle genome copy numbers were calculated by quantitative PCR targeting the vector transgene region. The rAAV2 vector used in this study was purchased as ready-to-use AAV2 particles from Addgene (Addgene viral prep #37825-AAV2).
Peipro transfection reagent
Manufactured by Polyplus Transfection
Sourced in France, United States
PEIpro is a transfection reagent for in vitro DNA delivery. It is a cationic polymer that complexes with nucleic acids and facilitates their uptake into cells. PEIpro is designed for efficient transfection of a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (2 mentions)
Optima xpn 100 ultracentrifuge, by Beckman Coulter (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
293ft cell, by Thermo Fisher Scientific (1 mentions)
Phelper, by Agilent Technologies (1 mentions)
Pwpi lentiviral vector, by Addgene (1 mentions)
Polybrene, by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Igg elution buffer, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 media, by Thermo Fisher Scientific (1 mentions)
Hitrap columns, by Cytiva (1 mentions)
0.45 μm filter flasks, by Merck Group (1 mentions)
Amicon ultra centrifugation filter, by Merck Group (1 mentions)
Puc57 2019 ncov s human, by GenScript (1 mentions)
Cellfectin 2 reagent, by Thermo Fisher Scientific (1 mentions)
Pvsv g vector, by Takara Bio (1 mentions)
12 protocols using peipro transfection reagent
1
Generating rAAV Particles in HEK 293T Cells
2
SFTSV M Segment Pseudovirus Production
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The open reading frame of SFTSV M segment, tagged with a C-terminal Flag-peptide (DYKDDDDK), was synthesized by GenScript Biotech (China) and subcloned into pcDNA3.1(+) vector. The N914Q mutant was constructed using the Q5® Site-Directed Mutagenesis Kit (New England Biolab, USA). The WT and mutant pseudoviruses were prepared as previously described62 (link). Briefly, HEK293T/17 cells were co-transfected with pLentiCMV-Luc2, pCMV-dR8.2-dvpr and the WT/mutant M expressing plasmids using PEIpro transfection reagent (Polyplus Transfection, France). The cell culture supernatant containing virions was collected at 48 h post transfection, which was clarified by low-speed centrifugation (1000 × g, 10 min) and filtered with a 0.45-μm cut-off syringe filter to remove the cell debris. The cleared supernatant was then layered onto a 20% (wt/vol) sucrose cushion and purified by ultracentrifugation (SW41 rotor, 209,900 × g, 2 h, 4 °C) using an Optima XPN-100 ultracentrifuge (Beckman Coulter). The resulting pellet was resuspended in sterilized 1×PBS (pH 7.4) and was used for subsequent Western blotting and infection assays. To quantify the viral titer, equal volumes of WT/mutant pseudovirus solutions were used to infect HEK293 cells. At 48 hpi, luciferase activities were determined using the Bright-Lumi™ Firefly Luciferase Assay Kit (Beyotime, China).
3
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells (ATCC® CRL-3216™) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were adapted to grow in suspension as described in [70 ]. The cells were cultivated in BalanCD® HEK293 medium (FUJIFILM IrvineScientific, Santa Ana, CA, USA) supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific™, Waltham, MA, USA) and maintained at 37 °C in an incubator with a humidified atmosphere of 8% CO2 in the air. Cell concentration and viability were assessed by the trypan blue exclusion method. LVs were produced by transient transfection using pALD-Lenti System (Aldevron®, Fargo, ND, USA) including the pALD-VSV-G-K, pALD-GagPol-K, pALD-Rev-K, and pALD-Lenti-EGFP-K plasmids. Briefly, the cells were transfected at 2.5 × 106 cells/mL in a shake flask using linear 25 kDa polyethyleneimine, PEIpro® Transfection Reagent (Polyplus-transfection®, Illkirch-Graffenstaden, France) at a mass ratio of 1:3 (DNA:PEI) and 1 µg of total DNA per 1 × 106 cells. At 48 h-post-transfection, VSVG-pseudotyped LVs (VSVG-LVs) were harvested at a cell density ranging from 4–6 × 106 cells/mL.
4
Generation of AAV Viral Vectors
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AAV viral vectors were generated using the methods of Challis et al. 91 (link). Briefly, 293FT cells (ThermoFisher Scientific) were grown to ~90% confluency in Corning hyperflasks (Corning) and co-transfected with 129 µg pHELPER (Agilent), 238 µg rep-cap plasmid encoding AAV5 capsid proteins (pAAV2/5 was a gift from Melina Fan (Addgene plasmid # 104964 ; http://n2t.net/addgene:104964 ; RRID:Addgene_104964) and 64.6 µg of either the CalEx plasmid (pZac2.1-GfaABC1D-HA-hPMCA2w/b) or control (pZac2.1-GfaABC1D-HA-hPMCA2w/b (E457A)) using the PEIpro transfection reagent (Polyplus). The AAV viral vector purification, concentration and titer determination was performed as previously described91 (link)–93 (link).
5
Expression of SARS-CoV-2 Spike and Membrane Proteins
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Synthetic cDNA with human codon optimization to express SARS-CoV-2 spike and membrane proteins were purchased from GenScript and cloned into the expression plasmid pcDNA3.1. Plasmid DNA was transfected using PEIpro® transfection reagent (Cat#101000017, Polyplus Transfection, New York, NY, USA) for Western blot and immunofluorescence analyses, as described previously [10 (link)].
6
Lentiviral Transduction of E1A Minigene
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The minigene reporter encoding the adenovirus E1A transcript was subcloned from pMTE1A plasmid32 (link),33 (link) into the PmeI site of the pWPI lentiviral vector (Addgene; Trono lab). Lentiviral particles were produced in HEK293T cells grown in DMEM medium with 10% FBS. The cells were transfected with pWPI-E1A and the packaging plasmids psPAX2 and pMD2.G (Addgene) using PEIpro transfection reagent (Polyplus transfection). After 54 h, the medium containing lentiviral particles was centrifuged at 1000 × g for 5 min and cleared using a 0.45 μm filter. The lentivirus was concentrated using Amicon Ultra 100 kD MWCO centrifugal filter units (Millipore) and aliquots were stored at −80 °C. For lentiviral transduction, cells were seeded in six-well plates with 2 ml medium and infected with the concentrated lentivirus in the presence of 8 µg/ml polybrene (Sigma). After 24 h, the culture medium was refreshed, and 36 h later cells were washed with PBS and harvested.
7
SARS-CoV-2 Virus-Like Particle Production
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HEK293T cells in a 10-cm dish were cotransfected with the plasmids encoding the SARS-CoV-2 M, E, N, and S or its mutants. In the transfection mixtures, 6 μg of each plasmid M-IRES-E, pcDNA3.1-SARS-CoV-2-N-Myc, and pCMV-3×Flag-S or the plasmid with S mutant were added into 1 mL of Opti-MEM and then mixed with 18 μL of PEIpro transfection reagent (Polyplus-transfection, France). The transfection mixtures were incubated for 15 min at room temperature and dropped into HEK293T cells. At 72 h posttransfection, the culture medium was collected, filtered through a 0.45-μm filter, layered onto 20% (wt/vol) sucrose cushions, and purified and concentrated by ultracentrifugation (35,000 rpm, 2 h, 4°C) using an Optima XPN-100 ultracentrifuge (Beckman Coulter) as described above. Moreover, the pellets containing VLPs were resuspended in sterilized 1× PBS, aliquoted, and stored at −80°C freezer.
8
Recombinant Production of ITS01 Antibody
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Production of recombinant ITS01 was performed as previously described (12 (link)). Briefly, HEK293T cells in 175 mm plates were transfected with 60 μg total DNA/plate at 80% confluency with PEIpro transfection reagent (Polyplus). Cells were co-transfected with the AAV ITS01 transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 16 hrs post-transfection, 10% FBS-DMEM media was replaced with serum-free 293 Freestyle media (Invitrogen). Media was collected after 48 hrs, debris was cleared by centrifugation for 10 min at 1,500 g and filtered using 0.45 μm filter flasks (Millipore Sigma). Proteins were isolated with HiTrap columns (Cytiva) and eluted with IgG Elution Buffer (Thermo Scientific) into 1M Tris-HCl Buffer, pH 9.0 (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1-2 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma). Heavy and light chains of the antibodies were assessed by Coomassie-stained SDS-PAGE. Antibodies were stored at 4°C before use and frozen at -80C for long term storage.
9
Production of SARS-CoV-2 Pseudotyped Viruses
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Pseudotyped SARS-CoV-2 was prepared as previously described [57 (link)]. Briefly, the plasmid pUC57-2019-nCoV-S (Human), containing synthetic cDNA to express SARS-CoV-2 spike protein with human codon optimization, was purchased from GenScript Japan Inc. (Tokyo, Japan) and cloned into the expression plasmid pcDNA3.1. Mutant spike cDNAs were synthesized using GenScript. The plasmids used in this study are listed in Supplementary Table S1 . For retrovirus-based pseudotyped virus production, HEK293T cells were co-transfected with spike-expressing plasmids containing phCMV-Gag-Pol 5349 and reporter pTG-Luc126 plasmids using the PEIpro® transfection reagent (Polyplus Transfection, NY, USA). Briefly, 2 × 106 293T cells were seeded in a T-25 flask on day 1, and the cells were co-transfected following the manufacturer’s instructions on day 2. On day 3, the growth medium was added to the flask for an additional two days of culture. The cell supernatant containing pseudotyped virus was collected, filtered through a 0.45 μm filter, and aliquoted to be stored at −80 °C.
10
Retroviral and Lentiviral Particle Production
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To produce retroviral particles, GP-293 packaging cells were transfected with the corresponding pLNCX2 plasmid and the pVSV-g vector (Clontech) using PEIpro Transfection reagent (PolyPlus, France). After 48 h, viral supernatant was then filtered and used to transduce target cells. Bacmids, obtained after transformation of DH10Bac bacteria (Life Technologies), were transfected into Sf9 cells (Novagen) by the Cellfectin-II reagent (ThermoFisher) to produce P1 baculoviral stock. P2 stock, obtained by infecting fresh Sf9 cells, was then titrated by qPCR and target cells were transduced at MOI = 500.
Lentiviruses encoding shRNA, siRNAs and controls were purchased from Dharmacon (Lafayette, USA). Target cells were transduced at MOI = 1.
Cells stably expressing the construct were selected by G418 (400 µg/ml) or puromycin (1 µg/ml) for 7 days.
Lentiviruses encoding shRNA, siRNAs and controls were purchased from Dharmacon (Lafayette, USA). Target cells were transduced at MOI = 1.
Cells stably expressing the construct were selected by G418 (400 µg/ml) or puromycin (1 µg/ml) for 7 days.
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