Jul 10, 2024
Lentivirus production and transduction
- 1Duke University
Document Citation: Shiyi Wang 2024. Lentivirus production and transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69jj1lqe/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: July 10, 2024
Document Integer ID: 82324
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Lentivirus production and transduction
1. Lentiviruses containing shRNA targeting vectors were produced to test the knockdown efficiency of shRNA constructs in cultured primary astrocytes or to bulk transduce neurons with shRNA and GFP.
2. To produce lentivirus, HEK293T cells were transfected with a pLKO.1 shRNA Puro targeting plasmid (for astrocyte transduction), an envelope plasmid (VSVG), and a packaging plasmid (dR8.91) using X-tremeGENE (Roche).
3. One day after transfection, the media was replaced with AGM (for astrocyte transduction), and media containing lentivirus was collected on days 2 and 3 post-transfection.
4. To assess the knockdown efficiency of shRNAs in astrocytes, rat primary astrocytes at DIV 7 were plated in 6-well dishes in 2 ml of AGM.
5. On DIV 8, 1 ml of AGM was removed, and 500 μl of fresh AGM was added along with 500 μl of lentivirus-containing media and 1 μg/ml polybrene.
6. Cultured astrocytes were treated with puromycin (1 μg/ml) from DIV 10-15 to select for transduced cells.
7. Cultured astrocytes were lysed at DIV 15 for protein extraction and Western blot analysis.