Help: Multichains stable transfection

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Sep 16 18:48:36 EST 1998


In article <2.2f.32.19980916165952.0067dae4 at osiris.univ-rouen.fr>,
inserm-u295 at UNIV-ROUEN.FR wrote:

> Hello,
> 
> I'm student and I'm currently working with a multichain protein (3 chains).
> In a first time, I would like to obtain stable clone with 2 chains of this
> protein stbly integrated but I have some problems.  Indeed, I obtained only
> stable clone for A or B, but no AB.  Each  chain is in SV40 vector (NeoR)
> and I made a cotransfection of A and B containing vectors. I use phosphate
> calcium transfection.   Has any body an idee to obtain an AB stable clone ?
> Is it possible to make multiselection with mammamlian cell (Zeo + Neo + Puro
> +...) without kill cells ?

You may want to consider a bicistronic vector to allow
simultaneous expression of both A and B from the same
promoter and with a single resistance gene.

If not you will need to use two different markers for A and B.  The
usual method is to transfect the cells sequentially but simultaneous
transfection should also be possible and I have also seen methods
where an "A" cell line and a "B" cell line are prepared and then
fused (this is good when you need to maintain the same point of
integration for single and double tranfectants).
     The cells are under a lot of stress when they are selected
and if your A and/or B protein is also toxic this will also cause
problems.  The cell will try and kick out one of the plasmids
or may even keep the plasmid but silence the expression of your
A or B protein.
     For best results you need to use different promoters for
the resistance genes and each of your proteins as otherwise
you may see one promoter silencing another.  This means that
you shouldn't run both A and B from the SV40 promoter.  There
is also a problem that the SV40 and CMV promoters show some
crosstalk.  An idealised setup would be;

Plasmid 1: neo gene driven by eg. TK promoter and gene A driven
by the SV40 promoter.

Plasmid 2: other resistance gene (pur, hyg, zeo etc.) driven by
the TK promoter and gene B driven by the RSV promoter.

Zeocin will probably not be an option as this is a relatively
inefficient resistance gene and requires a strong promoter
(usually CMV) so this will tie up one of the promoters.  I have
seen vectors with TK driving neo or hyg or pur.  You may want
to see how resistant your cells are as one antibiotic may be
considerably cheaper than the others.
     Good luck,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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