Jan 09, 2026

Public workspaceLentivirus production

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmb656g3p/v1
  • Akio Mori1,
  • Robert Edwards1
  • 1University of California San Francisco
Akio Mori
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmb656g3p/v1
Protocol Citation: Akio Mori, Robert Edwards 2026. Lentivirus production. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmb656g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 19, 2025
Last Modified: January 09, 2026
Protocol Integer ID: 233012
Keywords: production of lentivirus, lentivirus production, lentivirus production this protocol, lentivirus, primary neuronal culture, neuronal culture, cell
Abstract
This protocol describes the production of lentivirus in HEK293T cells for the transduction of primary neuronal cultures.
Materials
Cells
  • HEK293T cells (early passage) (CRL-3216, ATCC)

Plasmids

  • Gene of interest in lentiviral backbone
  • Accessory plasmids: pMDLG/pRRE, pIVS-VSVG, pRSV-Rev

Reagents

  • FuGENE transfection reagent (E2311, Promega)
  • Neurobasal medium (21103-049, Gibco)
  • Poly-L-lysine for coating 6-well plates (P2636, Sigma-Aldrich)

Consumables

  • 6-well tissue culture plates
  • 0.45 μm filter units
Troubleshooting
Cell seeding
Coat 6-well plates with poly-L-lysine for 1 h, and wash twice with PBS.
Seed early-passage (< P10) HEK293T cells onto coated 6-well plates to reach ~70–80% confluency the next day.
Transfection
Prepare a DNA mixture per well:
a. In 200 μL Opti-MEM, combine:
- 1.5 μg plasmid encoding the gene of interest (pFUGW, pJHUG, or pJHUIC)
- 0.5 μg each of accessory plasmids (pMDLG/pRRE, pIVS-VSVG, pRSV-Rev)
b. Add 12 μL FuGENE HD transfection reagent to the DNA solution.
c. Mix gently by vortexing.
d. Incubate at room temperature for 10–15 min to allow complex formation.
Remove HEK cells' medium and replace it with 1.8 mL of fresh medium per well.
Add the transfection mixture to each well and incubate overnight at 37°C, 5% CO₂.
Virus production
The next morning, remove the transfection medium and replace it with 2 mL neurobasal medium or HEK cells' medium per well.
Incubate cells for 36–48 h to allow virus production.
Collect the culture medium containing lentivirus. Filter the collected medium through a 0.45 μm filter to remove cellular debris. Use the viral supernatant immediately for neuronal transduction, or aliquot and store at -80°C.
Infection of primary neurons
Add 20–40 μL viral supernatant per cloning ring of primary neuronal culture to achieve efficient transduction.