In article <54qj36$fip at bignews.shef.ac.uk>,
Kevin Mulcahy <K.Mulcahy at sheffield.ac.uk> wrote:
>>Another possibility may be that the cells were not able to support the
>episomal maintainance of two different plasmids with the same origin of
>replication? Does naybody know if this is likely in mammalian cells?
I think this might be a problem, but I'm not positive. It shouldn't be a
*general* problem, but with the OriP/EBNA1 system I'm pretty sure it will
be. This system keeps low-level replication going, as I understand it,
and there aren't many copies of the episomes. This being the case,
there's only the selection to keep both plasmids going in the cell; if
there are very low copy numbers maintained then it may be difficult for
the cell to keep both plasmids going even in the presence of selection.
This may (again, I'm guessing) be exacerbated by the fact that COS are
monkey cells. I don't know if the OriP/EBNA1 system is as effective in
non-human primates. If you get even a small loss of efficiency in the
system then you may have a major loss of plasmid maintenance.
Brett Lindenbach said, " You might also consider utilizing the SV40 large
T antigen present in COS-7 cells to replicate your plasmid from an SV40
origin." If I understand your intention - to make stable cell lines -
this won't work. The SV40 ori drives unregulated replication, and the
plasmids driven by this reach enormous numbers in the cell - like 100,000
copies - which is enough to kill the cell. SV40 ori plasmids in COS
rarely last for more than a few days before the cells die off. (This
isn't something I've tested, though in transient transfection I do see the
COS starting to die off after a few days, but the texts say so.)
One other minor point is that the COS line I've been working with is
extremely resistant to hygromycin. What levels are you using, and are you
sure it's giving a good selection? It sounds like it is, from your post,
but it might be worth reviewing this.
This probably isn't much help, but otherwise I'm at a loss too. Good
luck. You might consider going with intergrating your plasmids rather
than episomal replication.
Ian York (iayork at 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