Lentivirus Production and Astrocyte Transduction
Shiyi Wang
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Lentivirus Production and Astrocyte Transduction
Steps
- Materials Required
- pLKO.1 shRNA Puro targeting plasmid (for astrocyte transduction)
- Envelope plasmid (VSVG)
- Packaging plasmid (dR8.91)
- HEK293T cells
- X-tremeGENE transfection reagent (Roche)
- Astrocyte growth media (AGM)
- Puromycin (for selection)
- Transfection of HEK293T Cells
- Plate HEK293T cells in appropriate culture vessels (e.g., T75 flask) to achieve 70-80% confluence on the day of transfection.
- Prepare transfection mix per flask:
- Combine 2 μg pLKO.1 shRNA Puro plasmid, 1.5 μg VSVG plasmid, and 1.5 μg dR8.91 plasmid in Opti-MEM.
- Add X-tremeGENE transfection reagent according to the manufacturer’s instructions.
- Incubate transfection mix at room temperature for 20 minutes.
- Add transfection mix dropwise to cells and gently swirl flask to mix. Incubate at 37°C with 5% CO2.
- Collection of Lentivirus
- Replace media with fresh AGM 24 hours post-transfection to enhance virus production.
- Collect lentivirus-containing media on days 2 and 3 post-transfection.
- Filter collected media through a 0.45 μm syringe filter to remove cell debris.
- Store lentivirus aliquots at -80°C for future use.
- Transduction of Rat Primary Astrocytes
- Day 7 In Vitro (DIV 7)
- Plate rat primary astrocytes in 6-well dishes at a density suitable for transduction experiments (e.g., 2 ml AGM per well).
- Day 8 In Vitro (DIV 8)
- Remove 1 ml of AGM from each well and replace with 500 μl fresh AGM + 500 μl lentivirus-containing media.
- Add 1 μg/ml polybrene to enhance transduction efficiency.
- Day 10-15 In Vitro (DIV 10-15)
- Treat transduced astrocytes with puromycin (1 μg/ml) to select for cells expressing the shRNA construct.
- Day 15 In Vitro (DIV 15)
- Lysate astrocytes to extract proteins for Western blot analysis to assess knockdown efficiency.
Notes:
- Handle lentiviruses in a biosafety level 2 (BSL-2) laboratory following institutional guidelines.
- Perform all steps involving lentivirus production under sterile conditions to prevent contamination.
- Optimize lentiviral titration to achieve desired transduction efficiency in astrocyte cultures.
- Ensure proper disposal of materials used for lentivirus production according to institutional biohazard waste disposal protocols.
