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.
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