Please help with stable transfection

John Ladasky ladasky at my-deja.com
Sat Oct 5 14:55:57 EST 2002


Khoa.Ngo at baker.edu.au ("Khoa Ngo") wrote in message news:<sd9c383e.051 at BAKER.EDU.AU>...
> Hi 
>   I am in the middle of the preparation of a stable transfectant clone
> with RAW264.7 murine macrophage cell line. For which, a source of about
> 50% positive stable transfectant cells was used for a limiting dilution
> with a ratio of 1/3 cell per well in a 96 well plate. Non-transfectant
> cells, used as supporter cells, were added together with the
> transfectant cells to the wells in medium without G418. After a few
> days, G418 was added to kill the supporter cells and in about 10 days,
> colonies were clearly seen under microscope for about 30 wells. From
> these, only 13 wells had colonies visuable to the naked human eyes, thus
> cells from each of the 13 wells were screened for positive clones.
> However, fluorescent microscopic observation (after immunofluorescence)
> shows some head-aching results for me. It is supposed to show either non
> or all cells are positive, but non of the clones could demonstrate a
> complete batch of positive cells, most of the clones have a few positive
> cells, while some show approx. 30 - 40 %  positive cells, which is less
> than the original source (a sample of the original source was screened
> to confirm this lesser effect).
> 
>      Here, i am wondering if someone can tell me what could have
> happened in order to lead to such results? and what would be the posible
> options available to get the expected clones? 
> 
>  I am really greatful for your response.
>  Many thanks in advance
>  Kind reagards
>  Khoa


Dear Mr. Ngo:

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.

Anoother poster suggested that your method of assessing the cells by
fluorescence microscopy might not be sensitive enough.  I do not think
that this is a big problem.  In addition to drug selection, I also
sort my highly-fluorescent cells with a FACS machine, which is quite
sensistive and quantitative.  Even this isn't enough to guarantee that
the cells that grow back will stay bright.

I cannot find any literature that discusses the issue that doesn't
come from a molecular biology company that wants to sell me something.
 However, from what I have been able to determine, the problem may be
due to the fact that most eukaryotic expression vectors in common use
express your gene of interest and the drug-resistance gene from
separate promoters.  When you select with drug, you are hoping that
the *close physical spacing* of the two genes on your vector will
prevent your gene of interest from being spliced away and lost through
recombination as the cells grow.  This close physical spacing clearly
helps, but I suspect that the linkage between your gene of interest
and the drug resistance gene is still significantly under 100%.  (If
you really want to struggle, try making stable transfectants by
co-transfecting two separate plasmids, one that expresses your gene of
interest and another that expresses your drug-resistance gene.  In my
hands, this approach is nearly hopeless.)

For decades, the dogma was that only bacteria posessed cistrons that
encoded for multiple proteins.  Recent research has shown that
multi-protein cistrons also exist in some eukaryotic viruses.  A
critical element known as an internal ribosomal entry sequence (IRES)
has been isolated from, for example, the encephalomyocarditis virus. 
The EMCV IRES is available in several eukaryotic expression vectors
now.  The IRES is not a promoter; it tells a ribosome that another
protein is encoded on an mRNA.  Place your gene of interest upstream
of the IRES, and your drug-resistance gene downstream.  The
drug-resistance protein can only be expressed if a) your gene of
interest is also expressed at the 5' end of the multi-protein mRNA
transcript, or b) the drug-resistance gene is somehow spliced out in
such a way that it lands right behind a promoter in the genome --
which is quite unlikely.

I have seen literature which suggests improved transfectant stability
with IRES vectors.  As I said, it is commercial literature.  I will be
giving an IRES vector a try for my next transfection project in a few
months.  You may want to investigate this option as well.

Good luck!

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



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