Sep 06, 2022
Lentivirus production
- Itika Saha1,
- F. Ulrich Hartl1,
- Mark S. Hipp2,3
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
- 2Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
- 3School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Protocol Citation: Itika Saha, F. Ulrich Hartl, Mark S. Hipp 2022. Lentivirus production. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4xn3gmk/v1
Manuscript citation:
The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
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: September 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69582
Keywords: ASAPCRN
Abstract
This protocol describes the production of lentiviruses to transduce HEK293T cells and has to be performed in a biosafety level 2 laboratory
Safety warnings
Has to be performed in a biosafety level 2 laboratory.
Lentivirus production
Lentivirus production
1d 1h 10m
1d 1h 10m
Plate ~3.6x106 Lenti-X HEK293T cells (Takara) in 10 cm dish in 10 mL standard DMEM. Cells should be ~80% confluent at the time of transfection.
Note
NOTE: Only low passage cells should be used.
Next day, remove 5 mL medium and replenish with fresh medium.
Warm up reduced serum medium e.g. Opti-MEM (Gibco) and transfection reagent to room temperature (RT). This protocol was performed with Lipofectamine 3000 transfection reagent (Thermo).
Add 24 µL Lipofectamine 3000 to 600 µL Opti-MEM, mix by vortexing and incubate 5 min at RT.
5m
In another tube, mix 6 µg plasmid containing gene of interest, 5 µg packaging plasmid psPAX2
(RRID:Addgene_12260), 1 µg envelope plasmid pMD2.G (RRID:Addgene_12259) and 24 µL P3000 reagent (provided by manufacturer along with Lipofectamine 3000 reagent) in 600 µL Opti-MEM, mix by vortexing and incubate 5 min at RT.
Mix contents of both tubes and incubate for 15 min at RT.
15m
Add DNA-lipid complex to cells dropwise.
2 days later, collect virus-containing medium and centrifuge for 5 min at 1,000 x g.
2d
Collect supernatant in a fresh tube and proceed with concentration.
Concentration
Concentration
1d 1h 10m
1d 1h 10m
Add Lenti-X concentrator (Takara) to clarified virus-containing medium at 1:4 dilution and mix well by gently inverting tube.
Incubate overnight or 2 h at 4 °C.
1d
Next day, centrifuge for 45 min at 1,500 x g at 4 °C followed by gently aspirating supernatant.
45m
Resuspend viral pellet in 100 - 1000 µL PBS, aliquot and store at -80 °C until use.