expression in ires containing vector

Aawara Chowdhury aawara at FEMA-trailer.org
Thu Jun 15 22:22:26 EST 2006


In <mailman.141.1150423724.17804.methods at net.bio.net>,
 Pow Joshi <pow.joshi at gmail.com> wrote:

> could anyone with the expertise in "IRES" expression vectors share
> with me their practical issues about the levels of expression of each
> of the cloned genes? I am trying to clone my insert of interest along
> with GFP as marker, and I am told that the levels of expression are'nt
> equivalent.
> Is there a way to overcome the problem?

No, there are too many variables - such as:
a) insert codon usage
b) insert sequence's influence on message stability
c) insert sequence altering message RNA structure, and thereby
   influencing the efficiency of IRES usage, or of translation

In general when we make cell-lines that stably express different
alleles (mutants) of the same protein, we screen multiple clones
for ones that express roughly equivalent levels of protein.  We
do this by quantitative immunoblot. 

Additionally, we've had the most success when using retroviral
vectors, where the resistance marker is expressed from the IRES,
and the introduced gene is expressed from the viral LTR. 

For human cell lines, our method of choice is to use episomal
oriP/EBNA1 vectors.  These work very well, and are not subject to
position effect variegation (PEV) that retroviruses (and
retroviral vectors) are subject to.  The only caveat with
oriP/EBNA1 vectors is that vector copy number per clone is
typically greater than 1 (anywhere from 4 - 10, in our
experience; others have reported as high as 40 - 50).  In our
experience, once a clone is picked, there is no further variation
of copy number within the clone - an observation consistent with
those published by Bill Sugden and co-workers.  Invitrogen sells
oriP/EBNA1 vectors, but you can also obtain them from Sugden's
group (ask for plasmids p205, p220, p294, or p1728). 

AC
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