picked GFP transfected colonies do not retain flouresence after being picked - (Jul/24/2006 )

I have CHO cells that have been transfected with pEGFP protein constructs (my protein of interest has been inserted into the pEGFPN1 and pEGFPC3 vectors). These cells have been in high conc. G418 media after transfection for a little over a month. I recently picked strongly flourescing colonies and put each colony into different wells of 6-well plates. Each colony successfully grew up in the new 6-well plates and formed healthy patches of cells, however none of the cells are flourescing...

Is there some reason for this?...Is there something I can do in the future?--Iam trying to make stably transfected cells through picking colonies and for some reason I can get cells to grow after one month in high conc. G418 but they do not seem to be stably transfected cells b/c they are not flourescing.

THanks
--Mini

Before drawing harsh conclusions, make sure that all your microscope lenses can transduce fluorescent light. I also had a sudden loss of fluorescence, but then I realized that my x20 lense transduces fluorescence effectively, but my x10 does not.
Maybe now that your cells grew you are looking at a different magnification...

If this is not the case then there could be other explanations -
( a ) Make sure that the cell line you started with did not have G418 resistence before you trasfected it, because if it did then you are not selecting for anything.
( b ) Maybe your protein is toxic to the cells and cannot be stably integrated. It such a case you can use a inducable promoter to express your protein only when you need it. However, since you said you saw GFP after a month I am not sure this is the case.

A good way to create stable cell line that is selected for high GFP, is to use FACS. Ask around and see if you have a FACS machine that can sort cells. If so, take your transfected pool and sort high fluoresent cells into a 96-well plate. It is essentialy the same as what you did, only more high-tech and it increases the number of positive colonies.

Thanks!

We do have FACS machine....will the sorted cells be able to be used for cell assays and time lapse movies (i.e. the FACS ensures sterile conditions such that these cells can be frozen and then used for different experiments)?

If you FACS is maintained well, it should be sterile. And after sorting you should be able to grow the cells, so you can start your experiments from homogenous clones.