I have an Env vector (5.5kb), Gag-pol vector (12.5 kb) and a gene of interest (8.7 kb) to be transfected using lipofectamine to 293T cells. According to the protocol of lipofectamine the concentration of DNA to be used is 500ng per well.
My question is, if I have three vectors of different sizes how should I calculate the concentration to be used? 5ug of 5.5kb vector carry more particles compared to 12.5 kb vector - how to calculate the concentration of these to bring equality for viral protein preparation? I am confused, please help. Protocol is attached for reference.
Dear Kavina,
For a 100 mm dish, I perform as the following ratio:
2nd gen: 10ug packaging (delta 8.2), 10ug transfer plasmid (pSPAX2), 5ug VSV-G (pMD2.G)
3rd gen: 5ug pGag-pol, 5ug pRev, 5ug VSV-G (pMD2.G), 10ug transfer plasmid.
Genemedi got a rich experience in lentivirus production, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
Kavina,
Is your 'gene of interest' something that will be packaged within the virus itself, such as a reporter gene? I'm working on the assumption it is for now...
For virus production stemming from the transfection of several individual viral components, I don't think you need to worry too much about exact concentrations in the manner you describe. It is more important to get the ratio of components right. For example, I recently produced MLV-based particles and transfected 293T cells with a [1.5 : 1.5 : 1] ratio of [Env : Reporter gene : Gag-pol]. The combined concentration of these plasmids should match the size of the cell culture vessel as well as the volume of transfection reagent you are using.
You mention wells, but for producing a virus stock (if this is indeed what you are doing) you may want to transfect a larger dish e.g. 10cm. The protocol for your transfection reagent will tell you how much DNA is needed for a dish this size, and then you can divide that figure up into the 1.5 : 1.5 : 1 components. So for example, if you need 12ug overall, you would transfect 4.5ug Env, 4.5 ug of your 'gene of interest'/reporter gene and 3ug of Gag-pol'.
Of course, the very first batch of virus you produce may not be perfect but you can hopefully produce something from which you will be able to devise a more precise protocol for your needs. Hope this is helpful.
Good luck!
I usually use 1 ug of your vector, 0.75 ug of psPAX2 and 0.25 ug of pMD2.G, per 1 million of cells.
Dear Kavina,
For a 100 mm dish, I perform as the following ratio:
2nd gen: 10ug packaging (delta 8.2), 10ug transfer plasmid (pSPAX2), 5ug VSV-G (pMD2.G)
3rd gen: 5ug pGag-pol, 5ug pRev, 5ug VSV-G (pMD2.G), 10ug transfer plasmid.
Genemedi got a rich experience in lentivirus production, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
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