one gene, two promoters

seppen at seppen at
Fri Dec 16 11:14:19 EST 1994

I am making a stable cell line expressing my protein of interest. (bilirubin
UDP-glucuronyltransferase) I am using a cotransfection protocol and select for
G418 resistance. The problem is that I do not obtain clones with a high
expression and only a low percentage of cotransfectants. (I use a 1:10 ratio of 
G418 resistance vector : cDNA vector) Another problem is that I cannot use a
viral promoter such as SV40 or CMV because I want to transplant the recombinant
cells in an animal and viral promoters are methylated and switched off once
inside an animal.
Now to my question, I have placed my construct under control of the PGK-1
promoter and want to clone this whole thing in the G418 resistance vector I
use. What will happen if I clone this downstream of the eukaryotic expression
promoter from this vector? (Rous Sarcoma Virus long terminal repeat promoter)
In other words, my gene of interest will then be under conterol of 2 different
promoters. Is this good or bad? I am really interested in comments of people
who have experience with 2 promoters upstream of 1 gene. Is only the promoter
closest to the gene active? The PGK-1 promoter is about 0.5 kb.
Any suggestions or comments....?
Thanks in advance

Jurgen Seppen
Academic Medical Center
Amsterdam, The Netherlands

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