I want to use HEK 293T cells for the production of lentiviral particles. I was told that after thawing the cells from the liquid nitrogen, I should split them 4 times in the presence of 1 mg/ml G418 before the actual transfection of the 3 plasmids (GAG, VSV-G, and carrier). Is this selection really necessary?
Yeah, I'd recommend the selection, to make sure you get the most of your experiments. You can split them with low dilution i.e. 1:3, so they' be confluent quickly again.
It's believed that the antibiotic treatment kills 293Ts that have silenced their large T expression. The large T helps replicate certain packaging plasmids that contain an SV40 origin and thereby presumably increase their yield.
I find the theory somewhat shaky, as the large T also provides a direct boost to cell replication speed. So anyone silencing it should be lost from the population, antibiotic or not.
I haven't seen any experiments asking your question directly, but it doesn't mean they're not out there. If anyone can post controlled experimental data about this, I'd be grateful too.
The only thing we can say for sure is that if you don't care to get the very highest yield you can get, it should be safe to proceed with packaging, see if you like the result. To be extra-safe, also consider doing the antibiotic treatment in parallel, so that you have something you can fall back to quickly, if you don't like the results of your packaging.
I never pretreat HEK293T cells for packaging lent-virus and I always succeed. But as John said, you might do it with or without pretreatment in parallel and see if you get any different results.
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