Stable transfection with or without EGFP - (Oct/26/2007 )

hi
i am new in the cell culture field so please who so ever know please help me
from last 6 month i am trying to get a stable clone , i am using HEK 293 cells which are already transfectd before and then i am inserting one more thing and i want stable transfection, i have clone my gene of interst in pEGFP c2 vector
so is it good to do stable transfection with EGFP or without EGFP
as many people told me when u use EGFP it makes too unstable and there is a chance that u loose ur gene of interset
please guide me
as my clone is showing flurocent but no sign of my gene of interset as i am checking it throuh elcetrophysiology
my gene of interset is approx 8 kb is it too long thats the reason cell might be thowing it out
please help me
thanks

I would make a stable cells with out the GFP unless its is absolutely necessary. Also I would verify the presence or absence of the protein by western before concluding that the cells are kicking out the gene.

I agree with scolix, if the EGFP is not necessary, please don't use it. You said they were transfected before you insert the gene. How long do you wait between transfection and inserting? We've had some problems with not waiting long enough in between (I think right now we wait 4 till 7 days) which made all the cells die very rapidly.
Other than that, just give it another try. Sometimes you can't really pinpoint what the problem is but it's just not happening. Good luck.