Need help with Blasticidin selection - (Mar/03/2010 )

My mammalian cell line of interest is already G418-resistant,so I did a co-transfection with my plasmid of interest (which will confer G418 resistence) along with pCMV-Bsd (will confer blasticidin resistence) at a 10:1 ratio (10ug and 1ug,respectively). Now I am selecting cells with blasticidin resistence (these cells should have taken up both plasmids during the transfection). However, when I was using G418+Blasticidin (2ug/ml) for selection,almost all cells died. So I am wondering if I should do another killing curve (because the G418+Blasticidin killing curve is different from the G418 curve or Blasticidin curve alone?). It seems Blasticidin doesn't work well for my cell line and it's not because my cell grow slowly in the presence of this drug--they just never grow. After 14 days of selection,the cells just died! I don't believe it's because none of them have taken up the pCMV-Bsd plasmid. Has anything similar happened to you guys?

Any input is welcome! thanks!


Grace

I suspect that the double insult of having G418 and blasticidin selection at one time is the problem. Do one of the plasmids first, select and once grown sufficiently switch it to a maintenance level (half to a 10th of the selective conc) of the selective agent, then transfect the second plasmid.