Transient vs stable transfection regulation?

agoodrid at vaxa.weeg.uiowa.edu agoodrid at vaxa.weeg.uiowa.edu
Fri Apr 9 14:12:35 EST 1993


In article: <1pqitq$f1o at news.ysu.edu>
ak949 at yfn.ysu.edu (Michael Holloway) writes:

>Does anyone know of an example of a reporter construct that can not be
>regulated in qualitatively the same manner in transient assays as in assays
>of a stably transfected line?  Or vice versa for that matter?

>I have a CAT reporter that I'm electroporating into PC12 cells.  I am
>utterly failing to reproduce results due to membrane depolarization published
>with a stable transfection of the same construct.  I'm mystified and wondering
>whether it's worth pursuing.  Any clues welcomed.


The differences in the method might be the cause.  Specifically, the
expression of the test promoter may vary depending on the average
concentration of the construct/cell.  In a stably transfected cell
the number of these promoters may be 1-50 but in transiently
transfected cells there may be thousands of these promoters/nucleus.
It is possible then that in transiently transfected cells that
transcription factors in low abundance may not be available for
a regulatory capacity that would occur with lower amounts of promoters.

I don't have any direct evidence for this (if someone does could you
post the reference).  I do know in some of our experiments that the
above "problem" explains some of the differences we see between
stably transfected cells and transiently transfected ones.

At one point we thought that the stably transfected clones had
an altered phenotype due to the pressure of selection or some other
reason.  This would mean that the response of the promoter differs
because of a change in one set of cells.  We tested this by doing
transient transfections of the stably transfected cells (You would
need a different reporter).  The result was the same as the other
transiently transfected cells indicating that the method of
transfection was the crucial variable.

There are a host of other reasons which are perhaps more likely.  The
above is one the other possibilities mentioned less often.
brianf at dna.uvm.edu (Brain Foley) writes:

>In my experience, stable clones vary greatly in their expression of
>reporter genes.  The reporter gene may become integrated into the host 
>genome in a different locus in each clone, thus leading to different
>expression controls in each.

Steve     AGOODRID at VAXA.WEEG.UIOWA.EDU



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