I want to do packaging in HEK293T by lentivairal vector 2ed and 3ed generation of lentivirus, but I do not know how much I should use each vector plus lipofectamine 3000.
For the second generation lentiviral vectors, I use 0.85µg of pLenti6 (lentiviral backbone vector), 0.5µg of pMD2-VSVG and 1.1µg of pCMV-R8.91 (contains gag, pol, tat and rev genes). With this mixture, I transfect 250 000 Hek 293T cells seeded in 10cm² dishes (~1welll of a 6-well plate) in 2ml of medium. I change the medium after 6 hours, and let the cells produce virus for 48 hours. Then I collect the HEKcell medium and filter it through a 0.22µm filter to remove any remaining HEK cells.
I add about 16µg/ml polybrene to the virus-containing medium, and use 1ml to transfect 1 well of a 6-well plate with target cells. The polybrene is not really necessary, but it improves the transduction efficiency. You can freeze the virus at -70°C for long time storage
Hello Maryam,
for 2nd generation lentis we use 3µg of each vector (expression + 2 x packaging) for 1-2x10^6 293T in a 10cm (diameter) dish. then proceed as above. So it depends on the number of cells you are going to transfect. Now you have a range using this and Sasker's Answer.
Good luck
p.s.: Freezing the particles may drastically (up to 50%) decrease virus titer - so if possible do transduction with fresh supernatant.
Hello Maryam
Last year I produced lentivirus in HEK293T, my conditions were:
- 6x10^5 cells/well (in a 6-well plate)
-7,5uL/well of lipofectamine with:
- pGag-pol: 1,2ug;
- pRev: 0,8ug;
- pVSV-G: 0,4ug;
- my plasmid: 1,6ug
Lentiviral particle-containing supernatants were collected 48 and 72 h after transfection, pooled and filtered using a 0.45-μm filter and stored at − 80 °C.
A Harvard protocol from Thomas Schurpf (Springer Lab) warns us that we should never use 0,22um filters, because the virus particles could be sheared and inactivated, therefore 0,45um filters should be used.
I hope this helps
André Simoes is right about the shearing, I also use 0.45µm filters, not 0.22µm filters.
Sorry, should have re-read my entry properly before submitting
How long is a piece of string? :)
There are so many protocols out there suggesting different amounts of plasmid! For a T-75 flask I use:
2nd gen: 10ug packaging (delta 8.2), 10ug transfer plasmid, 5ug VSV-G (pMD2.G)
3rd gen: 5ug rev (pRSV-rev), 5ug RRE (pMDL-RRE), 5ug VSV-G (pMD2.G), 10ug transfer plasmid.
Something worth keeping in mind is that if you are using a very large transfer plasmid, you may wish to increase the amount of that plasmid (as 10ug will result in proportionally less copies of the plasmid, if that makes sense)
Just to add too Jill's last point: In theory you want to use equimolar ratios of the three vectors. So the actual micrograms you have to use will differ with your vectors.
E.g. a (roughly) 1:1:1 Ratio could mean:
- 3µg pMD2 (5.8 kb)
- 6g of psPAX2 (10.7 kb)
- 10µg expression vector (15kb)
Good luck!
Also see here for very robust lentiviral protocols.
http://www.broadinstitute.org/rnai/public/resources/protocols
There are many answers because there are many solutions: It changes based on your transfection method, your shuttle vector and even based on your DNA prep. You should do a titration of the 3 (or 4) plasmids, starting with some of the protocols given here each time you change any DNA prep and find your best proportions. With LV you can go from very good transduction to nothing.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line