Please help with stable transfection

John Ladasky ladasky at my-deja.com
Wed Oct 9 16:39:36 EST 2002


Kyle Legate <legatek at mcmail.cis.mcmaster.ca> wrote in message news:<Pine.SOL.4.33.0210072011240.12951-100000 at mcmail.cis.mcmaster.ca>...
> On 5 Oct 2002, John Ladasky wrote:
> 
> > The problem of making stable transfectants that are truly stable is
> > unfortunately a common one.  Like you, I have had the percentage of
> > fluorescence-positive cells decline as I continue to grow cells.
> > Other researchers in the laboratories where I have worked have also
> > had trouble.  It doesn't always happen, but when it does it is
> > extremely frustrating.
> >
> Couldn't this arise from gene silencing? Although you have generated a
> stable transfectant, the cells may have figured out a way to shut off the
> gene of interest.

I suggested a separation of the drug resistance gene from the gene of
interest by recombination.  You are suggesting selective silencing as
another possibility.  I agree, it is possible.  An IRES-based
expression vector should address the problem in either situation,
though perhaps at the expense of getting a lower frequency of stable
transfectants in the initial round.

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
USA
Earth



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