Paul.xu at 18:31, 6 March 2021

2021-03-06T18:31:47Z

New page

packaging Plasmids:

psPAX2 503, -->Gag, Pol https://cdn.cellecta.com/Cellecta-psPAX2-in-CPCP-K2A-Map.pdf

pCMV-VSVG, pMD2.G, 505-->VSVG https://cdn.cellecta.com/Cellecta-pMD2.G-in-CPCP-K2A-Map.pdf

do not know if pRSV-Rev 504 is required

[https://manuals.cellecta.com/lentiviral-construct-packaging-and-transduction/v10a/en/topic/lentiviral-packaging-protocol https://manuals.cellecta.com/lentiviral-construct-packaging-and-transduction/v10a/en/topic/lentiviral-packaging-protocol]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830501/
The lentiviral transfer vector DNA, together with psPAX2 packaging and pMD2. G envelope plasmid DNA were combined at a ratio of 4:3:1, respectively. Production of 3rd generation lentivirus was performed using the combined ratio of transfer plasmid, packaging plasmid, Env plasmid and pRSV-Rev plasmid at 4:2:1:1,

Acrodisc purification
Purification of lentivirus using a Mustang Q Acrodisc (PALL) was performed according to the manufacturer’s recommendations. Briefly, 50 mL aliquots of lentivirus supernatant were adjusted to 25 mM Tris-HCl, pH 8.9 and loaded onto a Mustang Q Acrodisc and filtered through the Acrodisc syringe filter. The Acrodisc was eluted using a step gradient ranging from 0.3 to 1.5 M NaCl in 25 mM Tris-HCl, pH 8.5. Filtered supernatant was then added to 36 mL pollyallomer conical tubes and ultracentrifuged at 20,000 g for 90 min.

Optiprep purification
For concentration using Optiprep (Axis-shield), a 220 μL cushion of Optiprep was added to pollyallomer conical tubes and lentivirus-containing supernatant (30 mL) was gently layered into each tube taking care not to disturb the Optiprep. Samples were ultracentrifuged at 20,000 g for 90 min. Media above the media/Optiprep interface was carefully removed from each tube and discarded. The residual media containing lentivirus and Opitiprep was shaken at 200 rpm for 2 hrs in sealed tubes. The resulting mixture was pooled and centrifuged at 20,000 g for 24 hrs. Supernatant was discarded and the remaining pellet was resuspended in 300 μL of PBS, shaken overnight at 4°C, and then stored at -80°C until use.
[[Category:Virus]]

Lentivirus pseudovirus - Revision history

Paul.xu at 18:31, 6 March 2021