Hi,
I generally made LV in HEK293T cell lines by triple transfection. I am wondering whether the newly generated LV will transduce the packaging HEK293T. In this way I could exploit the transduced HEK293T as stable clone (after Puro selection) and thus speed up the process of stable clone generation. Someone tried this approach?
Hi,
Well why not but generally you want to know the titer of your lentivirus production to transduce your conditions with the same MOI.
Best,
Daniel
Hi, for your question, I never try to select 293T after the triple transfection even I had an idea like you. Because I obseved that 293T cells using for LV packing are so stress, and also not in good shape. I think that the cells even could be alive after puromycin selection but could not express your target gene well. So it is better to collect the virus particle then infect to fresh 293T.
Hi Dario!
Short answer, absolutely NO!
First, the cells are actively producing the virus at early stage, they are overly stressed and especially for good virus preparations, they won't survive long after viral harvest. The stable clones deriving from these cells will be severely altered and, even when surviving, won't be resembling anymore wild-type HEK293T.
The second reason is probably even more important, and it's safety. These cells have been exposed to packaging plasmids and lentiviral backbone. Given the high concentration of plasmids used, the plasmids can occasionally recombine or being randomly integrated into the genome, giving rise to cell lines that constitutively produces lentiviruses. In rare events, plasmids can recombine with each other, even reconstituting fully functional lentiviral particles (capable of replication). For these reason, packaging cells should always be inactivated after the last viral harvest (e.g. with bleach).
If you want to make stable HEK293T routinely during virus preparation, the simplest and safest thing is to infect a new well of wild-type HEK293T with a small fraction of your viral harvest. They are very easy to transduce and there's no reason for keeping the packaging line as a stable stock.
Hope it helps
Francesco
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