The concentration of lentiviral vectors by PEG-it ™ precipitation the PEG-it ™ Virus Precipitation Solution provides a simple and highly effective means of concentrating lentiviral particles. PEG is a polyethylene glycol formulation optimized for the precipitation of all lentiviral-based particles. Precipitation of the PEG-it virus the solution is a 5x solution.
- Transfer the supernatant to a sterile container and add 1 volume of cold PEG-it virus precipitation solution (4ºC) every 4 volumes of supernatant containing lentivector. (Example: 5ml PEG-it with 20ml of viral supernatant).
- Refrigerate overnight (at least 12 hours). Lentivector containing mixed supernatants with PEG-it virus precipitation the solution is stable up to 4-5 days at 4 ° C.
- Centrifuge the supernatant / PEG-it mixture at 1500 × g for 30 minutes at 4 ° C. After centrifugation, the Lentivector particles may appear as a beige or white pellet at the bottom of the boat.
- Transfer the supernatant to a new tube. Centrifuge the residual PEGit solution by centrifugation at 1500 × g for 5 minutes. Get rid of all traces of liquid by aspiration, being very careful not to disturb the lentiviral particles precipitated into granules.
- Resuspend / combine lentiviral granules in 1/10 to 1/100 of original volume using cold sterile phosphate-buffered saline (PBS) or DMEM containing 25 mM HEPES buffer at 4 ° C.
- Aliquot into cryogenic vials and store at -70 ° C until ready for use.
PEG-it protects the concentrated virus
The PEG-it virus not only concentrates its lentivirus preparations more than 100 times, but it also acts as a cryopreservative agent. PEG-it concentrated lentivirus lasts longer in the freezer and also survives quite well in aliquot freeze/thaw cycles. To test this, 50,000 HepG2 or HT1080 cells were seeded in 500 microliters of DMEM medium containing 10% FBS in 24-well plate formats. The next day, the medium was replaced with a fresh medium containing 1x TransDux Virus Transduction Reagent (Catalog # LV850A-1, System biosciences).
Frozen Aliquots of virus packaged with an MSCV-GFP-T2A-RFP expression cassette (catalog # LV605VA-1) were thawed on ice and 2 ul of Virus was added to each well in triplicate. The aliquots were immediately frozen in dry ice and thawed again in normal ice and 2 ul of the virus was again added to another three wells. The process was repeated three more times. Seventy-two hours after transductions, cells were imaged for green fluorescence and documented. The images below show how well PEG-it protected the virus from multiple freeze/thaw cycles and produced high levels of GFP and RFP virus transgene expression.
- Lentivector packing kits
Unique plasmid mixes that produce all necessary virus proteins and the envelope glycoprotein of vesicular VSV-G stomatitis virus required to produce active pseudo viral particles. Producer Cell Line 293TN (SBI Cat. # LV900A-1) temporarily transfected with packaging plasmids and an HIV base. The lentiviral construct produces packaged viral particles containing the lentiviral construct of interest.
- 293TN Human Kidney Producer Cell Line
(Cat. # LV900A1) For packaging plasmid lentivector constructs.
- UltraRapid Global Lentiviral Titration Kit
(Cat. # LV961A-1) Allows you to accurately measure the number of copies (MOI) of lentiviral constructs integrated into the genomic DNA of target cells after transduction with either SBI or HIV-based IVF lentivectors using sensitive qPCR.
- Cloning and expression lentivectors (many available)
Choose from shRNA, cDNA, or TRE based on FIV and HIV cloning and expression vectors.