Cells spontaneously zeocin resistant?

We've routinely made stable cell lines using plasmids containing
zeocin resistance. However, recently some of our starting cell lines
have apparently become spontaneously resistant to zeocin at fairly
high doses, at least 1000 ug/ml.

We've considered a couple of possible explanations.

(1) Have we mixed up cell lines so that we're accidentally growing a
resistant stable cell line? If so none of the markers that go in
with the resistance are present, including GFP (the resistance gene
we use is from Invitrogens pTracerCMV2 plasmids, which contains a
GFP/ZeoR fusion protein). We have also gone back to our earliest
freezes from liquid nitrogen without success. And at least two
rather different cell lines are behaving this way, even though the
two lines look and behave differently otherwise.

(2) Has our zeocin gone bad? It's relatively fresh and as far as I
can tell has been treated properly (i.e.e kept frozen). It kills
some other cell lines as expected at reasonable concentrations, 100
ug/ml. I am reluctant to spring for a new batch of zeocin if this is
still good, because it's rather expensive.

Are there other explanations? Could mycoplasma contamination cause
zeocin resistance?

Thanks,

Ian
--
Ian York ([Only registered users see links. ]) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England

In article <ftlijb$hm$[Only registered users see links. ]>, [Only registered users see links. ] (Ian A. York) wrote:

The whole cell lines became resistant or you start occasionally seeing
clones in the mock transformations?

DK

In article <J1yLj.53$[Only registered users see links. ]>,
DK <[Only registered users see links. ]> wrote:

Either the whole cell line, or at least a large fraction of the
cells. A well with zeo, seeded at about 10% confluence, becomes
confluent at roughly the same rate as an untreated well.

Ian
--
Ian York ([Only registered users see links. ]) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England

In article <fto04d$r3n$[Only registered users see links. ]>, [Only registered users see links. ] (Ian A. York) wrote:

Wow. I've never heard about such thing but mycoplasma
would be my only guess. New batch of serum, perhaps?

Certainly not bad zeocin since it kills other lines normally.

DK