expression construct question

arnaud.garcon at syngenta.com arnaud.garcon at syngenta.com
Fri May 26 03:33:36 EST 2006


Hi Becky,

You can do either. Invitrogen do a bicistronic vector (pBud something or
other). With it you get a first MCS under CMV promoter and a second one
under a hEF1 promoter. Works OK but is quite expensive,and uses zeocin
as a selectable marker which is crap and very expensive.  You can also
find vectors with one CMV promotor and 2 MCS separated  an IRES
(Internal ribosome something site) which is supposed to work in a
similar way as the pBud. Main problem with those is that you need a
fairly strong promoter for the 2nd site or you can see a lower
expression of the second gene compared to the first one. A pain if they
are supposed to form an active complex.
But at least pBud is pretty good for stable cell line generation as long
as you get a good handle as to how zeocin works.
Oh and I've heard that using 2 CMV promoters could be a problem as you
can get a recombination of the vector at the 2 CMV sites and excision of
one of your insert. So I've been told anyway.
 
I've grown out of those and clone my two genes on two separate vectors
(separately in Ecoli) each with a different selectable marker for
mammalian transformation (I use Hyg B and Puromycin as they are the
quickest working I've found). For expression you can either transform
them sequencially creating a stable cell line with one of your genes or
co-transform them with a double selection witch saves you a good few
weeks and works as well if not better as long as you can assay both
genes expression relatively easily.

Best of luck

A.- 


> -----Original Message-----
> From: methods-bounces at oat.bio.indiana.edu 
> [mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of 
> Rebecca Pickin
> Sent: 25 May 2006 14:59
> To: methods at magpie.bio.indiana.edu
> Subject: expression construct question
> 
> I am in the process of generating an expression construct 
> which will express 2 different cDNAs.  Would it be better to 
> use a vector which contains 2 mcs and 2 promoters (in 
> addition to antibiotic selection for E.
> coli and mammalian cells) or could I insert the cDNAs tandem 
> using a CMV promoter and BGH Poly A?  
> 
> Thank you!
> Becky
> 
> Rebecca Roche Pickin, PhD
> 
> Post Doctoral Associate
> Center For Environmental Health Sciences College of 
> Veterinary Medicine - Basic Sciences Mail Stop 6100 
> Mississippi State, MS 39762
> 
> 
> 
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> 



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