shRNA selection in suspension cell culture - (Dec/04/2009 )

I am transfecting shRNA into suspension cells. The shRNA has a turboGFP reporter and puromycin gene. How do I do selection in suspension cells? Which is a better selection method, using GFP reporter or using puromycin selection? How many days after transfection should the selection be done?

You can select with puromycin, treating the cells till your control cells (untransfected cells) are dead, then you can assume all the remaining cells in your transfected cells have the puromycin resistance. If you want to make stable cell line you have to keep the puromycin selection.
With the gfp you can sort the cells, and select the gfp+ ones.
I dont remember how many days do you need for puromycin, neither the concentration (it depends of your cell line) but normally in 4 days with the right concentration of puromycin (1-10ug/ml) the control cells should be dead.

But how do I pool out the live cells from the dead ones in suspension culture?

solcole on Dec 4 2009, 04:00 PM said:

Ok. Well, if you just select with puromycin you can pool them down at low rpm and dicard the supernatant. With the sorting you dont have any poblem I guess.

hmmm ok...let's say I FACS sort the GFP positive cells 2 days after transfection. How do I know if these GFP positive cells have the shRNA integrated into their genome (stable knockdown cells)?

So if you want to make stable cell lines you have to keep them in puromycin for a while to be sure that they dont lose the plasmid. The point is that if you do a sorting you are sure that your cells have the plasmid with gfp and the resistance, and then you can keep them. If you dont do the sorting you just have to keep them in puromycin for a while to be sure that all of them have the plasmid. To get rid of the death cells you just have to centrifuge them at lets say 1000 rpm, collect them, wash them if you want with pbs and put again in culture with medium plus puromycin. I would keep them in a maintenance concentration of puromycin

If you want to create stable's with transfection alone I'd just keep puromycin on for a week or more.
All the transiently transfected cells will hopefully have lost their cDNA's by then allowing only the stable cells to grow under puromycin.

The dead cells are a little bit of a problem though so sorting GFP+ after this period of puromycin incubation might be the "cleaner" option.

In general though, due to the low efficiency of creating stable clones this way, viral transduction is usually far superior, especially for suspension cells..