stable transfection high expression - problems with low expression in stably transfected cells (Sep/07/2007 )

Hi All

I would like to ask for some advice in generating stably transfected NIH 3T3 cell lines.

I have generated NIH 3T3 cells lines stably epxressing some constructs. I have been using the pZeoSV2+ vector and pcDNA3.1+. RT-PCR shows my fragment is transcribed but I cannot see my myc-tagged-protein in a western blot. I have tried making a transient transfection in which I was able to detect the protein although the signal was very weak. So I'm thinking the pZeoSV2+ and pcDNA3.1+ vectors are not the best. Which system do you use?. As some of my transcripts are supposed to work on the mRNA level I can't use viral transduction since I'm not interessted in my mRNA transcripts containing much more RNA than my fragment of interesest.

Please help!

Thanks

Louise

You can try and push expression of transgenes by incubating cells with Sodium butyrate, TSA or Azadeoxycytidine.