I am currently attempting to package lentivirus in COS-7 cells (I have ethical objections to 293 cells) and would welcome advice on how to proceed in my endeavor using Lipofectamine 2000 and a second generation lentiviral system in 6 well plates. I believe this will work due to the expression of SV40, but I do not want to poison my cells with an overdose of Lipofectamine 2000. I also have access to Lipofectamine LTX PLUS reagent if that would be less toxic.
COS-7 cells will work but the virus will be much much less than with 293T.
This gives you a distinct disadvantage if you want to transduce difficult cell types....you might have to upscale the transfection.
The other (cost) disadvantage is that 293T is easy to transfect with PEI to >90% while COS-7 is probably more like 40-50% so you will have to use the more expensive reagents.
Lipofectamine 2000 will work fine >90%, DNA at 3:1 ratio for COS-7. LTX plus also i suspect but i never used it.
transfect, change media after 24h and harvest after 48h (you can sterile filter through 0.45 if you are making a large scale )
I always found an ultracentrifuge best for concentration again large scale... depends what you have available 10,000rpm for 12-16h overnight at 4 degrees will also work.
Mary Kate Kilroy Hi! Have you obtained good results using this cell line or have you found a new one?
Hi Francesco,
I switched focus to another project, but I think COS-1 might work better. I am also looking into transposon based systems (PiggyBac) for gene delivery that eliminates the need for the packaging cells. COS7 have worked very well for our transient transfections based experiments though.
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