Jul 11, 2024
Lentivirus Production and Astrocyte Transduction
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Lentivirus Production and Astrocyte Transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1934klr2/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: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103193
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Lentivirus Production and Astrocyte Transduction
1. **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)
2. **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.
3. **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.
4. **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.