Replication-incompetent reporter virus stocks were generated with a co-transfection of a VSV-G-pseudotyping vector and a HIV-1 NL4-3 full molecular clone lacking Env with IRES-GFP inserted behind Nef at a ratio of 7.5:2.5. In brief, 10 μg of DNA total was transfected (PolyJet, SignaGen) into 5 × 106 HEK293T cells (ATCC, CRL-3216) according to the manufacturer’s protocol. In total, 25 ml of supernatant was collected at 48 and 72 h and combined. Virus-containing supernatant was filtered through 0.45 mm PVDF filters (Millipore) and virus precipitated in 8.5% polyethylene glycol (PEG, average Mn 6000, Sigma-Aldrich), 0.3 M sodium chloride for 4 h at 4 °C. The supernatants were centrifuged at 3,500 rpm for 20 min and virus resuspended in 0.5 ml PBS for a 100× effective concentration. Aliquots were stored at −80 °C until use.
0.45 mm pvdf filter
Manufactured by Merck Group
The 0.45-mm PVDF filter is a laboratory filtration device made of polyvinylidene fluoride (PVDF) material. It is designed to filter liquids or gases, retaining particles larger than 0.45 millimeters in size.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (4 mentions)
Pspax2, by Addgene (3 mentions)
Polyjet, by SignaGen (2 mentions)
Lenti x concentrator, by Takara Bio (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
100 mm cell culture dish, by Corning (1 mentions)
C2c12 cells, by Merck Group (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
D6750, by Merck Group (1 mentions)
Jetpei transfection reagent, by Qbiogene (1 mentions)
Hitrap, by Cytiva (1 mentions)
Retro x concentrator, by Takara Bio (1 mentions)
7 protocols using 0.45 mm pvdf filter
1
Lentiviral Vector Production for Transduction
2
Vpx-Containing Virus-Like Particles Production
3
Generating dCAS9-KRAB Lentiviral Particles
4
Lentiviral-mediated Gene Perturbation in C2C12 Cells
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Lentiviral particles were generated using HEK293T cells (ATCC). Cells were seeded at 50–60% confluency in 100 mm cell culture dish. The following day, cells were transfected with 5μg of the vector of interest (dCAS9-KRAB with specific sgRNAs), 1μg of pMD2.G and 4μg of psPAX2 (Addgene) using 30μL of Lipofectamine (2000). 24 h post-transfection the media was changed. The supernatant was collected 48hr post-transfection, filtered with a 0.45-mm PVDF filter (Millipore), and concentrated using the lenti-x concentrator (Takara, 631232) according to the manufacturer’s instructions. Concentrated viruses were stored at −80°C.
C2C12 cells were transduced in suspension and plated in DMEM medium supplemented with 6 μg polybrene. Cells were collected five days post-transduction and GFP positive cells were sorted for RNA extraction.
C2C12 cells were transduced in suspension and plated in DMEM medium supplemented with 6 μg polybrene. Cells were collected five days post-transduction and GFP positive cells were sorted for RNA extraction.
5
Production and Purification of Channelrhodopsin-2 AAV
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pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE was generously provided by Karl Deisseroth (Stanford Univ.). For preparation of AAV, HEK293T cells were grown in DMEM media with antibiotics and FBS. The day before transfection, four plates beyond 90% confluence from 10-cm dishes were plated onto five 15-cm dishes and incubated for 18–22 h or until 60 to 70% confluence. HEK293T cells were transfected with pAAV-DIO-ChR2-EYFP, pAAV-DJ and pHelper using jetPEI transfection reagent (QBiogene). The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 20 min. After incubation, the transfection mixture was added to each 15 cm dish. Transfected cells were harvested 48 h after transfection and incubated with 0.5% sodium deoxycholate (Sigma; D6750) and 50 units/ml of benzonase nuclease (Sigma; E1014) at 37°C for 1 h. After removing cellular debris by centrifuging at 3000 × g for 15 min, the supernatant was filtered through a 0.45 mm PVDF filter (Millipore). Purification of AAV- DJ particles was performed using HiTrap heparin affinity columns (GE Healthcare). For concentration of AAV, Amicon ultra-15 centrifugal filter units with a 100,000 molecular weight cutoff were used. Concentrated virus aliquoted and frozen for storage at −80°C. The final viral concentrations was 3~6 × 1012 virus particles per ml for each AAV.
6
Lentiviral-mediated Gene Perturbation in C2C12 Cells
7
Generating Retroviral DRReRNA Vectors
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