Apr 03, 2023
Virus production
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi 2023. Virus production. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jpkjl1b/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: April 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79877
Keywords: ASAPCRN
Abstract
Lentiviral production protocol
Virus production
Virus production
For lentiviral production, HEK cells were transfected with the plasmids expressing the shRNAs together with the lentiviral packaging plasmid psPAX2(RRID:Addgene_12260) and the envelope plasmid pMD2.G (RRID:Addgene_12259) using TransIT-X2 (Mirus).
HEK 293 T cells were seeded in a 10-cm dish to reach 80% confluency the day after
transfection.The next day, the medium was changed.
On days 4 and 5 after transfection, the medium was collected and filtered through a0.45-
μm PVDF membrane.
To concentrate the virus, the filtered supernatant was centrifuged in a Vivaspin column (Sartorius Stedim Biotech) at 3000 RCF at 4 °C until the volume reached 500
–1000μl.
The virus-containing supernatant was collected, and the concentration
of p24 particles was determined with Lenti-X GoStix Plus (Takara).
Cells were infected with equal concentrations of p24 particles for the
scramble and respective shRNA.