Stable Transfection of Cells in Suspension

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Sat Feb 26 06:39:33 EST 2000


Mikkel wrote:

> Hi !
>
> I have a problem regarding stable transfection of cells in
> suspension. I use the Tet-On system from clontech and my
> gene of interest is EGFRvIII. My problem is that every time
> i put a selction pressure on the transfected cells the
> non-transfected cells die (as they should) but the contents
> released from these cells kill the transfected ones.
> Does anyone know of an easy way of how to separate the dying
> cells from the living ?

Are you sure your hypothesis is right? I simply cannot imagine an experiment to check if a cell dies because of the selection pressure or because of
material released from other dying cells.
We´ve also been doing transfection of suspension cells (Jurkat T lymphocytes in our case), and have found that they are poorly transfected by most methods.
The few cells that ARE transfected (i.e. show up green when expressing EGFP) are then subject to selection. As always, only a very small percentage of
transfected cells will actually become "stably transfected", whereas most transfected cells simply die because they do not integrate the plasmid into the
genome. I guess what you see is simply cell death by selection. If your transfection efficiency is low, there is very little chance to get a good number of
stables.
Try electroporation, it works well for transfecting difficult cell lines.

As to the separation of dead cells from live ones, this is hardly possible with suspension cells. One way to improve the ratio of live vs. dead cells is
split them quite early after transfection to have only very small number of cells in each well (microtiter plate). The live ones will propagate, and will
form clones.

Hope this helps,
Frank







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