expression construct question

Jayakumar, R R.Jayakumar at roswellpark.org
Thu May 25 15:20:27 EST 2006


Which mammallian expression vector has two MCSs with two promoters???
Even then, you may have problem getting stable expression of both cell
lines.  The right thing would be to select stable transformant with
first cDNA construct, and then transfect the stable transfectant with
the second construct carrying the second cDNA and select for stable
transfection (provided you are doing transfection).  Or perhaps you
could replace the GFP gene in some vectors like pBIEGFP (clontech) with
your second cDNA construct.  That has a bidrectional promoter which is a
great idea, though both sides do not express uniformly in my experience.

   I believe each cDNA should have its own PolyA signal.  Except for
mitochondrial compartmentt, I am not aware of the nuclear genome
expressing polycistronic RNAs.
   Best of luck
Jai
 

-----Original Message-----
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Rebecca Pickin
Sent: Thursday, May 25, 2006 9:59 AM
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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