Double-transfection (stable) of COS-7 cells

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Sat Oct 26 14:18:02 EST 1996


On 26 Oct 1996 14:59:25 -0400, Ian A. York <iayork at panix.com> wrote:


>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.  

Your reasoning is correct.  If the hygromycin selection is not effective,
the rate of loss of that plasmid will be different than the rate of loss
the neomycin plasmid.  

>I've no idea how long "multiple generations" would be.  

Rates of loss for oriP plasmids and their derivatives have been published
by Bill Sugden and his co-workers.  One such recent reference is listed
below.

----------------------------------------------------------------------
UI  - 95115089
AU  - Kirchmaier AL
AU  - Sugden B
IN  - McArdle Laboratory for Cancer Research, University of Wisconsin
      Medical School, Madison 53706.
TI  - Plasmid maintenance of derivatives of oriP of Epstein-Barr virus.
SO  - Journal of Virology 1995 Feb;69(2):1280-3
AB  - oriP is the origin of plasmid replication of Epstein-Barr virus.
      Replication from oriP requires both the cis-acting elements (the
      family of repeats and the dyad symmetry element) and the viral
      origin-binding protein, EBNA-1. The ability of plasmids containing
      oriP to be maintained stably in EBNA-1-positive cells reflects the
      efficiency both of their replication and of their segregation each
      cell cycle. The efficiency of plasmid maintenance was determined for
      plasmids containing derivatives of oriP with one copy of the dyad
      symmetry element and two copies of the family of repeats by
      measuring the rate at which they were lost from cells in the absence
      of selection. These measurements demonstrated that plasmids with
      derivatives of oriP with two copies of the family of repeats in one
      orientation are maintained only slightly less efficiently than is
      wild-type oriP. To determine whether plasmid maintenance could be
      affected by reinitiation at the dyad symmetry element (T. A. Gahn
      and C. L. Schildkraut, Cell 58:527-535, 1989), plasmids containing
      derivatives of oriP with two copies of the dyad symmetry element and
      one copy of the family of repeats were compared with plasmids
      containing wild-type oriP in EBNA-1-positive cells. These
      measurements showed that plasmids containing a derivative of oriP
      with two copies of the dyad symmetry element are maintained as
      efficiently as is wild-type oriP and are not amplified relative to
      wild-type oriP. These observations indicate that the trans-acting
      factors that regulate DNA to replicate once per S phase are
      insensitive to multiple cis-acting regulatory sites within a
      replicon.
----------------------------------------------------------------------


There is one other point I wish to emphasize.  There is no arbitrary
"maximum"  copy number of oriP plasmids that can be maintained within cells.
The number of oriP plasmids within any particular clone is HIGHLY
dependant upon the number of copies of the oriP plasmid initially 
introduced into the cell, i.e the efficiency of transfection.

later,
Ashok

-- 
Ashok Aiyar, Ph.D.
Department of Oncology            email: aiyar at ebv.oncology.wisc.edu
University of Wisconsin-Madison                  tel: (608) 262-6697




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