Double-transfection (stable) of COS-7 cells

Ian A. York iayork at panix.com
Sat Oct 26 13:59:25 EST 1996


In article <slrn574a2v.i7n.aiyar at ebv.oncology.wisc.edu>,
Ashok Aiyar <aiyar at ebv.oncology.wisc.edu> wrote:
>On 26 Oct 1996 10:47:30 -0400, Ian A. York <iayork at panix.com> wrote:
>>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.  
>
>This is not a problem with oriP containing origins.

In the short term (several generations) I agree; but over many
generations, I think it might become a problem.  Here's my reasoning:

The OriP/EBNA1 system leads to the stable maintenance and the regulated
segregation of the episome at relatively low levels.  If there are two
oriP-bearing episomes, the total count of oriP in the cell will be roughly
the same, which means that each episome will be at around half the
concentration compared to a single episome.

With each cell division, the episomes will be replicated and sorted
between the daughter cells.  Again, the individual episomes won't be
sorted, the total oriP-bearing population will.  That, combined with the
relatively lower levels of each episome, might mean that purely
stochastically over multiple generations one episome will become less
abundant than the other.  That difference will be magnified and eventually
you'll end up with a line that only has a single episome.  

If the cells are under strong selection, then those expressing only one
episome won't grow and will never become a problem.  If the selection is
not stringent, however, the population as a whole may hvae a large number
of cells only bearing one episome or the other.  

I've no idea how long "multiple generations" would be.  I suspect that if
you're cloning the transfected cells, then the number of generations for a
single cell to reach a usable volume (if you go with as few as 10^5 you're
looking at, ummm, 20 generations or so, right?) might be enough for a
problem.

I'm just guessing on this, and if oriP has some way of distinguishing
individual episomes - I can't think of a simple way offhand, but EBV is
way smarter than I am - then this is all irrelevant.

Ian

-- 
      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



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