IRES-Element

Emmanuel Skoufos skoufs at minerva.cis.yale.edu
Tue Jul 9 15:38:00 EST 1996


On 8 Jul 1996, Matthias Vogel wrote:

> Dear Netters!
> 
> We are searching for a well characterized IRES-Eelement. Which IRES 
> should we use? We need it for expression in lymphoid cell lines.

I think that the poliovirus or the EMCV IRESs are the best characterized
so far and they seem to work in various cell lines
 
> Is it better to place the resistance marker in front of the second 
> reading frame or the other way around?

I think that the resistance marker should be the *second* cistron, just 
in case your element does not function on the particular cells.  If the 
marker is expressed (selection), then you know that your IRES is 
functional in the live cells
.
If it is the first (cap-dependently expressed) cistron you will not be 
able to know whether or not the gene in the second cistron is expressed, 
unless you have an assay for it (Abs, etc).  I do not think that there is 
a report out there about this, but I think that cap-dependent translation 
might give you more protein than cap-independent under "normal 
circumstances"  ( i.e. cells not infected with picornaviruses). If you 
know of such experiments please let me know:-). You want your expressed 
gene and not the marker to be expressed under optimal conditions.

> Is it possible to express three proteins with two IRES-elements in between?

In principle it should work (bacteria do it all the time :-) ). In practice, 
IMHO such vectors will be too large to tranfect into most cell types, but 
it would be an interesting experiment to do with a minimal expression vector.

Emmanuel



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