antisense oligomers

[37_410 at uwovax.uwo.ca]

In article <1991Jun7.170303.8279 at mbcf.stjude.org>, bugg at mbcf.stjude.org writes:
> Does anyone out there have any experience with antisense oligomers?  We have
> this idea to try and prevent translation of our protein of interest with an 
> antisense oligomer overlapping the 5' end of the gene.  According to what 
> literature we can find on the subject, it is important to try to stop 
> translation near the start codon, because if you let the protein get started, 
> the oligomer will not prevent its formation.  Are there any methods for getting
> the oligo into the cells other than passive diffusion?  Has anyone out there 
> tried delivering a plasmid which produces antisense RNA into a cell to turn off
> a gene?
> All advice on the subject would be appreciated.  Thanks!
> 
> Barbara Bugg  BUGG at ST.JUDE.ORG
> St. Jude Children's Research Hospital
> Memphis, Tennessee  USA

I don't know if this reply went through the first time so I've  re-sent it.

We have done some preliminary attempts at producing antisense
RNA  in order to limit the synthesis of a gene for one of the subunits
of the E. coli RNA polymerase subunits.  We cloned  antisense
fragments onto multicopy plasmids such that expression of the
antisense  RNA was under the control of the tac promoter.  Thus
we would induce their expression  in transformants of the plasmid
via the addition of  IPTG.  None of our fragments appeared to work,
however none of them actually overlapped the 5' end of the gene
for reasons relevant  to that genes regulation.
Thus it appears that the 5' end may be important in this case as in
most if not all procaryotic cases.  In eucaryotes  (as far as I have
read) the 5' end is not  always as critical.  Hlowever eucaryotes
appear to have other fancy tricks such as "unwinding activities".
I don't know whether this info helps or not but I thought  I'd post it
anyway.
			Best wishes
				Lou Passador.