Loss of plasmid from Cos-1 cells

wetsel_r at msnews.wustl.edu wetsel_r at msnews.wustl.edu
Wed Dec 8 23:38:08 EST 1993


In a previous article, drg at prophet.pharm.pitt.edu (Duke Groebe) wrote:

>Description: Re: Loss of plasmid from Cos-1 cells
> 
>I've transfected COS-1 cells with a plasmid carrying both a gene under CMV
>control and neomycin resistance.  Since the transfection was performed 
with
>ccc DNA, I presume the plasmid is carried episomally.  Cells selected 
under
>G418 pressure were then plated (approx. 10% confluent) in meduim lacking
>G418.  At near-confluency I assayed for the expressed gene.  My question
>is:  How quickly will the plasmid be lost from the dividing cells in the
>absence of selective pressure?

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
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