Rearranging plasmids, which strain to use

Gabor Keresztes g.keresztes at removethistoreply.uni-bonn.de
Fri Mar 26 13:58:03 EST 1999


Dear netters,

    My aim is to express two similar proteins (wt and mutant) in
eukaryotic cells. To this aim I chose the pIRES vector which enables the
expression of two genes from one plasmid.  However, when trying to put
the second insert into the vector the resulting clones will invariably
(16 out of 16 prescreened by PCR)  be rearranged by the time I grow up
the bacteria (strain DH5alpha)  for minipreps. This was forseeable to
some extent, as the plasmid is relatively large and the tandem repeat of
the genes (1.3 kB each) provides a nice target for recombination.

    Some companies claim to have strains (Sure from Stratagene or Stabl2
from Gibco) which are supposed to circumvent my problem. Does anyone
have any experience as to which strain is the best. Stratagene itself
claims that Sure is capable of maintaining 98% of the plasmids without
rearranging at one cell cycle. However, by the time one grows up a
maxiprep even that is not too promising as in 30-40 cycles roughly half
of the plasmids will be already rearranged and my experimental system
would be severly affected by the expressed rearranged molecules. Before
ordering any of these strains I would appriciate comments on that.

    I would also appriciate comments as to how stable such a vector
could be in eukaryotic (cos) cells.

    Or should I straigh away forget the idea of using a bicistronic
vector??



                                        Gabor Keresztes, Ph.D.
                          Section of immunobiology,University of Bonn




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