Stable cell lines not overexpressing my gene of interest!
Posted 08 January 2005 - 11:09 AM
I am trying to make stable cell lines (in NIH3T3) overexpressing my gene of interest. I did a quick screening (using RT-PCR) and found the 'stably' transfected cell lines did not have the transfected plasmid DNA integrated in the genome.
I am using mammalian expression vector pCl-Neo (Promega) and G418 as the selection media. The problem I am encounting did not seem to be a transfection problem because one of the 'stable' cell line was the 'vector only' and that seemed to work fine (i.e. I was able to amplify a region within the vector using cDNA made from this 'vector only stable cell line).
Any suggestions/ideas would be very appreciated (I am just a bit worried that the same problem would occur if I repeat the stable transfection again!)
Posted 10 January 2005 - 04:56 PM
I find that with almost any stable selection I will have to select and screen a lot of stable colonies in order to find one that expresses my protein at the level I want (if at all). For what ever reason cells are good at keeping the parts of the vector they NEED to survive and disposing of those they don't, particularly if those parts have them doing extra work like over-expressing a "useless" protein.
As for your situation you have to llok at the obvious: is your protein likely to be in any way toxic to the cells? If it has been expressed before by others ten it is at least not totally lethal to the cells but if it is a unique expression then only trying will tell it it's toxic. If it is you will need to do inducible expression.
Can you easily try other expression vectors? I have not had good luck with the proteins I tried to express with pCI-neo (just my personal experience) You may want to try other vectors. I know pCDNA3.1 is an old vector but I have yet to have it fail in my hands. You could also try IRIS vectors though they are hit and miss in my hands as well.
Have you screened enough individual colonies? I mean like a hundred?
Have you checked transient expression levels of the protein 48-72 hours after transfection? If you cannot get transient transfection you cannot get stable.
Sorry if most of this is redundant, I hope it helps.
Posted 11 January 2005 - 02:28 PM
I have looked at transient expression levels and they seemed to be OK.
I think I haven't screened through enough clones (far less than hundreds!!) I will do that straight away before I chuck away my cells!