Loss of plasmid from Cos-1 cells

Duke Groebe drg at prophet.pharm.pitt.edu
Fri Dec 10 12:13:44 EST 1993


In article <8DEC93.23380837 at msnews.wustl.edu>, wetsel_r at msnews.wustl.edu
wrote: 
> Duke:
> 
> Let me toss in my $0.02 worth.  Your vector sounds like one of the 
> InVitrogen vectors, as that's what we're using.  We've presumed that the 
> plasmid is integrated into the cells DNA as we've obtained a positive 
> signal on genomic southerns of the transfectants.  I may be mistaken but I 
> presumed that they are episomal for a short period of time until they 
> either a) kick it out, or b) integrate it under selective pressure.
> 
> Question:  ccc DNA???  What's the "ccc"?
> 
> We cloned the bulk culture of transfectants by limiting dilution and 
> selected for a) high copy number by genomic southern and b) expression of 
> protein - if possible.  We carried many of these transfectants for a while, 
> 
> like about 6-8 months before observing the expression of our protein 
> beginning to decrease with time (assayed by immunoprecipitation).  
> Presuming that revertants had occurred and were taking over the culture, 
> G418 was re-applied to no avail.  99% of cultures (as best can be seen 
> visually) were G418 resistant over two weeks.  This is the first instance
> where we thought the cells may have kept the G418 marker and jettisoned the
> transfected cDNA (subjective speculation!). 
> 
> Second, I've been trying to get a quick PCR of my transfectant up and 
> running so that I can determine the presence of my cDNA *prior* to going 
> through the effort of limiting dilution, sorting, etc.  I have a large cDNA 
> 
> (5Kb) that I've tried to put into COS-1 cells (via Dotap, or CaP04).  In
> this case too, the cells suffer some mortality and endure G418 selection. 
> However, upon repeated PCR analysis, the cells do not carry my cDNA as far
> as I can tell but are G418 resistant.  A second instance where the cells 
> have tossed the cDNA in lieu of G418 resistance.
> 
> To your question, after selection and subcloning, they should (under score 
> *should*) be usable for what you need for quite some time, presuming they 
> have met your initial criteria for expression.  In our case, after G418 
> selection we were doing immunoprecipitations for many months on our 
> transfected cells it was only after long term culture (6 months to a year) 
> when we noticed the production of "our favorite protein" dropping off with 
> time.
> 
> I hope this helps,
> David
> 

Thanks.

I haven't gone to nearly as much trouble as you have to confirm the
presence or absence of my DNA in COS-1 cells (By the way, cccDNA is
"covalently-closed, circular DNA".  I thought that it was important to
imply that I was not selecting for stable transformants (a linearized
vector would greatly facilitate genomic integration) but rather was hoping
to get the cells to keep the plasmid replicating as a separte but necessary
(i.e. G418 resistance) entity).

You're correct in assuming that I am using a vector from InVitrogen
(pcDNA3) which carries a CMV promoter to drive expression of the cloned
gene and an SV40 promoter to drive neomycin resistance and allow
intracellular plasmid replication.

As it turns out, I got some (not a lot) expression of my gene (alpha7
subunit of the nicotinic acetylchoine receptor) as measured by radiolabel
binding to G418-selected cells grown in the absence of G418.  In an awkward
development, cells grown in the presence of G418 did not show ANY
detectable radiolabel binding.

Since I want to do mucho mutagenesis, I don't want to have to select for
stable transformants every time and would rather have a quick and efficient
method (transient expression would be best, episomal next best) for
screening "interesting" mutants.  Since my assay is radiolabel binding
(required for the drug I am investigating), I need a good signal/noise
ratio.  I get lousy sig/noise ratios (but measurable) for transient
expression in COS-1, HEK 293, and NIH/3T3 cells.

Barring wierd stuff like cell-culture temperature, my current conclusion is
that expression of this particular protein is not good, period, in
transfected cell lines.  I don't anticipate that stable transformants would
provide me with any greater levels of expression.  Ergo, I'm outta here
(Hmmm, wait, patch-clamp?).

I attended a seminar which plugged InVitrogen's pcDNA3 (FASEB 1993) and the
speaker (Dr. Sandra Day?) said that 6 months is about the time cells have
for episomal expression before the "load" of carrying the plasmid takes its
toll.  After that, they conk out.  Looks like your data confirms that.

Duke



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