bugg@mbcf.stjude.org (06/08/91)
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@ST.JUDE.ORG St. Jude Children's Research Hospital Memphis, Tennessee USA
37_410@uwovax.uwo.ca (06/11/91)
In article <1991Jun7.170303.8279@mbcf.stjude.org>, bugg@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@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.