Hi experts,
I am trying to produce lentivirus/AAV-producing HEK293/FT? I have a few questions regarding the protocol and results. I followed multiple sources, but the one I really followed is from Addgene (https://www.addgene.org/protocols/), with a few modification. I did not use the chemical-based transfection rather the electroporation method that I established. My questions are:
1. When I perform initial (GFP-containing plasmid DNA) transfection to HEK293, isn't it that cells harboring the GFP-containing plasmid will fluoresce in two scenarios:
a. HEK cells was successfully transfected with all three plasmids (1) transfer plasmid (which contains GFP/gene-of-interest), envelope plasmid and packaging plasmid, and
b. HEK cells was successfully transfected with the "transfer plasmid" ONLY, meaning without envelope and packaging plasmids.
2. Given that in (1), both scenarios are possible, how can I really isolate and generate lentiviral particles? I have already performed ultra-centrifugation of filtered supernatants from cell at post-transfection (72-96 h), however I did not get a good amount, or when I transduced the concentrated "lentiviral particle" I did not see any GFP-expressing cells (successful transduction).
Are there any protocol that may give me a step-by-step guide to lentiviral production? I appreciate the help in advance! Thanks.
Hi.
You can follow this routine lentivirus infection protocol to establish your HEK293 stable cell line.
https://www.abmgood.com/Documents/files/Full-A4-lenti-infection.pdf
Good Luck!
I haven't worked with the lentiviral particles you have, but from what I've seen, usually the issue is just with transfection efficiency. If you're trying to transfect three plasmids at once, it can be pretty stressful on the cells, and efficiency is low if you don't use a complex condenser. 293T is easy to transfect, but I would still venture to use a cell-line specific transfection reagent (https://altogen.com/product/293t17-transfection-reagent-emb-kidney-crl-11268/) and that might help you get larger amounts of lentiviral production.
Hi Ted,
Right now, I am accustomed to electroporation and lipofectamine 3000 methods. I will definitely look into that but from my posting, my concern is that 3 plasmids at once or just 1 (transfer) plasmid can be transfected at the same time. So there's no way of knowing if the packaging was successful until I go to next step, which is harvesting the (lentiviral) particle. I just wonder if there's a unique step to ensure that the lentiviral particle is packaged, or at least improve its packaging.
And theoretically speaking, transfection reagents like lipofectamine 3000, FuGENE and, even, the one you sent shouldn't really make any substantial difference to the transfection method.
When I see Addgene's protocol, they use polyethyenimine (PEI) and Chloroquine diphosphate, which are just transfection reagents. I am not sure if there's any special property that ensures the transfection of 3 plasmids in a cell. But empirically speaking, I will always get the same result: 3 plasmids transfected and 1 plasmid transfected showing the fluorescence.
Thanks,
Edward
Hey Edward,
I think that if you're following Addgene's protocol then you should use the transfection methods that they did (electroporation was not one of them). The electroporation buffer or transfection reagent that you use does make a big difference in context of the cell line (reagents that work in HEK293 will have far lower efficiencies in N2a), but most reagents are tested on HEK293, so you're right that it probably won't make that big of a difference in your case.
I would also read this paper (Article Production of lentiviral vectors
) which explains a lot about plasmid packaging and methods of lentiviral production. In particular, the authors note that "for small-scale applications, other very efficient cationic transfection agents including lipofectamine, fugene or 293fectin, have also been used resulting in high expression levels." In your case, substituting PEI for lipofection is likely to bring beneficial effects, but because HEK293 is an adherent cell line, using electroporation will be detrimental to overall efficiency (electroporation is far better suited for suspension cells).With regards to a "special step" for guaranteeing lentiviral production, I'm not sure one really exists at this point. Usually researchers use equal molar quantities of plasmids, and then some of the resulting virions/proteins will form into functioning lentiviral particles. I guess more research is needed, but I wouldn't be the one to ask about completely novel approaches for lentiviral production.
I hope you find a working way to generate lentiviral vectors. Good luck!
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