Ecdysone inducible system HELP - (Jan/11/2006 )
Hello there -
New to this board, but I am hoping I can get some feedback...
We use the ecdysone inducible system from Invitrogen in our laboratory. We have had the system almost since its introduction and have made numerous mammalian cell lines (mostly based on 293T) using this inducible promoter and VgRXR.
Here is my problem and it's potentially a big one - When we archive these cell lines (by freezing in N2 liquid), after thawing, we are finding that the inducibility of the cell lines is, in many cases, drastically reduced! We use the lines to produce virus stocks and proteins and we simply cannot get the cells to work properly after freezing and thawing. They are still resistant to every antibiotic present on the plasmids having to do with the ecdysone system and I have even subcloned one line to see if I could re-find a clone that had the original inducibility, but to no avail.
Has ANYONE ever heard of this happening with this system or know of anyone who has used it? We have been wracking our brains trying to figure out what is happening...any ideas?? We are tapped out!!
Thanks everybody...
MLWC
i am just try to test the ECR systerm very soon. I will let you know when I am done
New to this board, but I am hoping I can get some feedback...
We use the ecdysone inducible system from Invitrogen in our laboratory. We have had the system almost since its introduction and have made numerous mammalian cell lines (mostly based on 293T) using this inducible promoter and VgRXR.
Here is my problem and it's potentially a big one - When we archive these cell lines (by freezing in N2 liquid), after thawing, we are finding that the inducibility of the cell lines is, in many cases, drastically reduced! We use the lines to produce virus stocks and proteins and we simply cannot get the cells to work properly after freezing and thawing. They are still resistant to every antibiotic present on the plasmids having to do with the ecdysone system and I have even subcloned one line to see if I could re-find a clone that had the original inducibility, but to no avail.
Has ANYONE ever heard of this happening with this system or know of anyone who has used it? We have been wracking our brains trying to figure out what is happening...any ideas?? We are tapped out!!
Thanks everybody...
MLWC
the same thing happened to me. by boss got angry because he could not believe this. i gave up ultimately. good luck!