DsRed stable transfections? - problems with Stoplight vector in Flip-in cells (Nov/03/2008 )

Hi,

I am have trouble with getting stable expression of DsRed in HEK cells. I am using the pStoplight Vector. which express DsRed until it is cut out by Cre Recombinase, and then GFP is expressed. I have cloned this into the Flp-In kit from Invitrogen to generate a stable cell line using flp recombinase.

It looks good for a while, but then, even though I have cells surviving the selection agent, after a week or so, I dont have any more red fluorescent cells!

So, I have no idea whats happening. It seems to me that either: 1) the transient expression of flp recombinase is somehow acting on all the stoplight transfected cells, somehow removing the gene... unlikely. 2) DsRed expression is somehow being driven down by the cell? 3) DsRed is somehow toxic and kills the stable cells, but then why does anything survive!?

Anyway, any suggestions or advise would be very much appreciated!

I am guessing that you have just had a transient transfection; I don't think FLP recombinase is the same as Cre recombinase. Cells will survive in the antibiotic quite well if the plasmid is being expressed transiently. Make sure you titrate your antibiotic before transfection.

The vector I am using has flp and cre recombination sites; flp to integrate and cre to excise. I titrated before, and I am using enough hygromycin to kill untransfected cells in about 3-5 days, and most cells do dies. After 2 weeks I see colonies forming from the cells that do survive. But they dont express the gene, DsRed, but they must be expressing the resistance gene...

I'm a bit confused and am not sure I fully understand exactly what you are doing but I've used the Flp-In system to create multiple stable lines in CHO cells. Am I correct that you are subcloning the DsRed into the pcDNA5/FRT/TO vector and then co-transforming the FRT vector and pOG44 recombinase vector? I had this happen once when I tried to make a GFP-tagged stable cell line. I didn't have any trouble making the GFP control cell line but my specific construct was toxic and killed cells over time (even with the tet-control). I would see the GFP-induced expression for about two weeks and then it was gone. I'm not sure why you would be having trouble with DsRed alone, especially since there are publications using this tag in stable cells but it sounds exactly like the situation I had.

Thats it exactly! its just DsRed, so it should not be toxic, and thats all that is expressed. It doesnt make any sense. But I definitely have colonies form that are not red.

I was wondering if the HEK cells could somehow turnoff the expression of DsRed, but not the resistance gene??