Sep 06, 2022
Lentivirus production for primary neuron transduction
- Miguel Da Silva Padilha1,
- Irina Dudanova1,
- Itika Saha2,
- F. Ulrich Hartl2,
- Mark S. Hipp3,4
- 1Molecular Neurodegeneration Group, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany;
- 2Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
- 3Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
- 4School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Protocol Citation: Miguel Da Silva Padilha, Irina Dudanova, Itika Saha, F. Ulrich Hartl, Mark S. Hipp 2022. Lentivirus production for primary neuron transduction. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39eo1g25/v1
Manuscript citation:
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: 69592
Keywords: ASAPCRN
Abstract
This protocol describes the production of lentiviruses to transduce mouse primary neurons and has to be performed in a biosafety level 2 laboratory
Safety warnings
This protocol describes the production of lentiviruses to transduce mouse primary neurons and has to be performed in a biosafety level 2 laboratory
Expand HEK293T cells (LentiX 293T cell line, Takara) for lentiviral packaging to 70-85% confluency in DMEM Glutamax (+4.5 g/L D-glucose, - pyruvate) supplemented with 10% Fetal Bovine Serum (FBS)(Sigma), 1% G418 (Gibco), 1% Non-Essential Amino Acids (Thermo Fisher) and 1% HEPES (Biomol).
Note
NOTE: Only low passage cells should be used.
For lentiviral production, plate cells (~ 4.8 x 106 ) in a three-layered 525 cm2 flask (Falcon).
On the following day, transfect cells with 59.52 μg expression plasmid, 35.2 μg packaging plasmid psPAX2 (RRID:Addgene_12260) and 20.48 μg envelope plasmid pVsVg (gift from Dieter Edbauer) using 345.6 μl TransIT-Lenti transfection reagent (Mirus) in 9.6 mL DMEM without FBS.
1d
Incubate transfection mix for 20 min at room temperature and exchange the cell medium in the meantime.
20m
Add 10 mL of transfection mix to the flask, followed by incubation overnight.
Exchange the medium on the following day.
1d
After 48-52 h, collect culture medium containing the viral particles and centrifuge for 10 min at 1,200 x g.
2d
Filter the supernatant through 0.45 μm pore size filters using 50 mL syringes and add 20 mL Lenti-X concentrator (Takara) to filtered supernatant.
Incubate overnight at 4 °C and centrifuge samples at 1,500 x g for 45 min at 4 °C.
1d
Remove the supernatant and resuspend the lentivirus pellet in 150 μL TBS-5 buffer (50 mM Tris-HCl pH 7.8, 130 mM NaCl, 10 mM KCl, 5 mM MgCl2).
After aliquoting, store lentivirus at -80 °C.
Thaw virus preparation immediately before adding to freshly prepared neuronal culture medium.
Remove a fifth of the medium from cultured neurons and add the equivalent volume of virus-containing medium.
Note
NOTE: Volume of concentrated virus to be added to the neurons and the length of transduction should be determined empirically.