Do you mean that you want to keep passaging cells that are already transfected and producing virus? Or are you asking if you can passage the 293T cell line for making virus in the future?

You can keep passaging transfected cells to produce more virus, but eventually the transfected plasmids will degrade. 

The 293T cell line can be passaged many times and still produce virus fine. My cells are up to about passage 100 and they're still producing the same titres they did 6 months ago.

Thanks Jill..!  Yes, I was asking about the already transfected 293T cells.  

Being little more greedy.. Do you have any reference showing that passaging of transfected 293T can be done (or has been done) with some selection marker eg. Puromycin ? 

Oh right, I understand your question now. The transfected cells seem to stop producing virus about 96h post transfection (I think I have a reference for this somewhere, but it's easy to measure). So there's no advantage to keeping the transfected cells going.

Dear Tanay,

We used to passage 293T cells many times, but have changed our policy on this. We bought fresh commercial stocks, expanded, subaliquoted and created a large bank which we froze. We thawed and batch tested to ensure consistency. We now only keep cells for a few passages before discarding them and thawing a new aliquot. We do this to minimise risk of infection contamination, and also because in our hands titres would slowly drop. It sounds like Jill is lucker (or better) than us.

Secondly, in terms of keeping transfected cells, one has to bear in mind that expression of fusogenic envelope glycoproteins (e.g. VSV-G) is cytotoxic.

Best wishes,

Simon 

I did forget that, when you express VSV-G in 293Ts it can cause the cells to fuse. Fine for virus production but not for their long term survival I guess.

Simon can I ask how much your titres are dropping over time? I'd like to think my consistent titres are because I'm awesome :) but I'm wondering if I just got a freakishly good aliquot of cells orginally...

Hi Jill, I'm hand-waving a bit here, because we started our current regime quite a while back, but I seem to remember that titres would fall to a quarter of their early passage values after the cells were P30 or so. It always used to puzzle us, as the cells looked very healthy, and definitely myco -ve, but just didn't want to make good vector any more. BW, Simon

Thanks Simon!

you can expand or passage transfected 293T cells to produce more virus in the future. This is a common strategy to scale up virus production for various applications, such as gene delivery, viral vector production, or viral-based assays. Here's how you can do it:

By expanding transfected 293T cells and optimizing virus production conditions, you can scale up virus production to obtain larger quantities of virus for future experiments or applications.

l With this protocol list, we might find more ways to solve this problem.