In article <U10784.9.000FEAF0 at UICVM>, U10784 at UICVM (Victor Levenson) wrote:
Hello Dr Levenson,
It seems that I am lucky today. In fact I was preparing digestions to
construct a polycistronic expression K7 for cftr cDNA, GFP cDNA and
beta-gal, spaced by IRES sequences, to be used under CMV promoter control,
And I have read your posting.
For the design of the above mentionned K7 I was based on Zitvogel L. et al
(Hum Gene Ther 5:1493-1506 (1994)) paper, where there is no discussion
about any particular positioning of the cDNAs and the IRES.
Do you think I have to review my construction before continuing and in
that case could you give me some more details about the rules to observe?
your advice will be very helpful for us
Molecular Pathology and Gene Therapy / University of Bordeaux II
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: IRES (internal ribosomal ENTRY site) or CITE (cap-independent translation
: enhancer) - containing RNA fragment assumes a specific conformation which
: allows ribosomal entry. Several papers have been published on the use of this
: sequence for simultaneous expression of two (or more!!) proteins using one
: promoter with several genes "linked" by IRESs (I can find the references, if
: you want). IRES is used in pCITE series of plasmids made by Novagen (no
:: According to the literature it is an effective way to ensure simultaneous
: expression of 2 or more proteins in the same cells. Beware, though, that ATG
: codon of your gene has to be in-frame with the rest of IRES and at a certain
: distance from the core structure; e.g. with pCITE2 it should be within NcoI
: site (ccatgg), the 1st cite in the polylinker. This limitation makes it
: cumbersome to clone genes under CITE.
:: If you are interested in details e-mail me.
:: Victor Levenson
University of Bordeaux II
Medical Biochemistry and Molecular Biology laboratory