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Bicistronic vector

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Thu Sep 17 20:57:11 EST 1998


In article <2.2f.32.19980917113055.006762ac at osiris.univ-rouen.fr>,
inserm-u295 at UNIV-ROUEN.FR wrote:

> I would like to obtain a  stable clone for 2 chains of a protein, but I have
> some problems.
> Bernard Murray suggest me, with other possibilities, to use bicistronic
vector.
> 
> Do you think that it is possible to obtain A and B chains with the following
> vector (if it is possible for me to construct it !) :
> 
> ....---pCMV--A chain--IVS--IRES--B chain--pA---....---pSV40--NeoR--pA--...
>

As I have mentioned, the CMV and SV40 promoters may interfere with
each other so that you end up with neo+ cells but no A or B expression
(this *can* happen).  Maybe substitute TK or RSV for SV40.
 
[snip]

> Thank you in advance for your help.
> P.S. : Thank you very much Bernard for your very interesting answer !!

You are very welcome...

If you look at eg. Clontech's pIRES1neo or pIRES1hyg vectors

they have [CMV]-[Multicloning site]-[IRES]-[resistance gene]

What it may be possible to do is to put the A chain in the MCS
and then swap the B chain for the resistance gene.  You could
then cotransform cells with this and eg. pTK-Hyg or pTK-neo.
This would mean that you don't have to work with one big plasmid.

A more elegant alternative is to grab the neo-R gene from
Stratagene's pBK-CMV or pBK-RSV and use this to replace
the ampR gene in the above (IRES1) construct.  Stratagene
have added a TK promoter to the neo-R gene which already has
the original (Tn5?) bacterial promoter so this confers neo-R
in mammalian cells and kan-R in bacterial cells.  This will
keep the size of an all-in-one construct within reasonable
bounds.
     You will end up with;

-[CMV]-[A]-[IRES]-[B]-[pA] ... [TK]/[Tn5P]-[neoR]-[pA]-

which should be less than 5 kb without the A and B genes.
     Before you embark on such an adventure it may be worth
checking that you see good expression of both A and B in an
IRES vector as sometimes the IRES does not function very
efficiently.
     Bonne chance!
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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