Cells spontaneously zeocin resistant?

[Ian A. York]

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   (iayork from panix.com)  <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