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Lentivirus Packaging & Harvest
Lentiviral packaging and harvest is the process of converting a DNA plasmid into infectious viral particles using a “producer” cell line. The LentiMax™ system (often associated with high-efficiency mixes from suppliers like SignaGen) optimizes this by providing pre-mixed packaging plasmids and specialized transfection reagents to maximize viral titer.
1. Packaging: The Transfection Phase
In the LentiMax™ system, HEK293T cells act as the “factory.” These cells are chosen because they express the SV40 Large T-antigen, which allows for episomal replication of plasmids and high protein production.
- Cell Seeding: HEK293T cells are seeded 18–24 hours prior to reaching 80–90% confluency.
- Complex Formation: The LentiMax™ Packaging Mix (containing Gag-Pol, Rev, and VSV-G envelope plasmids) is mixed with your Transfer Plasmid (containing your Gene of Interest) and a transfection reagent like CalFectin™.
- Transfection: The DNA-reagent complexes are added dropwise to the cells. The cells begin transcribing the viral components and assembling particles.
2. Harvest: Collecting the Virus
The virus is secreted into the cell culture supernatant. Harvesting typically occurs in two “waves” to maximize yield:
- First Harvest (24–48 hours post-transfection): The culture medium is collected and replaced with fresh medium.
- Second Harvest (72 hours post-transfection): The culture medium is collected and replaced with fresh medium. This represents the peak of viral production.
- Third Harvest (96 hours post-transfection): The final supernatant is collected. Beyond 96 hours, HEK293T health declines, increasing cellular debris and decreasing titer.
- Clarification: The collected supernatant is centrifuged (e.g., 500 x g for 5 minutes) and usually filtered through a 0.45 µm low-protein binding filter to remove dead cells and debris.
Summary Table: LentiMax™ Workflow
| Step | Timing | Key Action |
| Seeding | Day 0 | Seed HEK293T at ~80% confluency. |
| Transfection | Day 1 | Add LentiMax™ Mix + Transfer Plasmid. |
| Media Change | Day 1 (+6 ~ 18h) | Replace transfection media with fresh growth media. |
| Harvest 1 | Day 2 (48h) | Collect supernatant; store at 4°C. |
| Harvest 2 | Day 3 (72h) | 2nd collection; store at 4°C. |
| Harvest 3 | Day 4 (96h) | Final collection: pool 3 harvests |
| Storage | Post-Harvest | Aliquot and snap-freeze at -80°C. |
Please contact us at info@signagen.com to discuss your lentivirus project
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