T7 polymerase stable expression in eukaryotic cells?

[Jack M. Bernstein, M.D.]

In article <1995Feb6.211136.28822 at oxvaxd> springer at immsvr.jr2.ox.ac.uk. (springer) writes:
>Subject: T7 polymerase stable expression in eukaryotic cells?
>From: springer at immsvr.jr2.ox.ac.uk. (springer)
>Date: 6 Feb 95 21:11:35 GMT

>Does anybody know about a vector that will stably express T7 polymerase
>in human or mouse cells, to a fairly high level? This is to put a gene
>of interest into these cells to be expressed from a T7 promoter (safety
>considerations), either by vaccinia or by transient transfection.

>Of course, we could clone the gene ourselves in pcDNA3... :-) 

>please e-mail: sebastian.springer at clinical-medicine.oxford.ac.uk

>Thanks!

The most widely used system would be the vaccinia-T7 system, which can be 
obtained from Bernie Moss at the NIH (vaccinia with a T7 gene cloned in 
expressing at high levels after infection of the cells with vaccinia). This 
works wonderfully well insofar as getting high levels of expression of many 
genes. Two caveats: 1. you obviously need a T7 promoter (found in pGEM and 
many commercial plasmids) 2. Putting an IRES site in just upstream of the 
start codon tremendously enhances expression/ translation.