stable transfection: need of picking single colonies - (Jan/03/2005 )

in my last lab, i was told never to use stable lines if i want to compare expression levels of different mutants. the reason for that was that different clones from even same mutant can have totally tifferent expression levels due to their integration sites at the genome.

so far.....

in my NEW lab, we do not pick single colonies while establisthing stable lines and take ALL cells that survive selection medium. so, i was told, we generate a kind of average expression level (because we have strong and weak expressing clones in that population). and because this average expression level, we can use stable cell lines to compare expression levels of tifferent protein mutants.


what do you think about that "average expression thing"?

It *seems* that the everage thing could have so many variables due to individual DNA prep quality, tranfection efficiency, tranfectant cell viability, etc etc, that you would be hard pressed to control for all of the variables in your experiment to say that it was the mutation that was making X% change in expression. Maybe simply a ton of repetitions with various DNA preps and different transfection reagents with the same results could get someone to believe but I might not hang my paper on that evidence alone.

What type of promoter are you driving it from: endogenous or forgein?