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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

-jensen-

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


QUOTE (jensen @ Sep 7 2007, 11:53 AM)
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

-LeserattePD-