2 replies to this topic

[biobio]

Hello all,

I have made some lentiviral vectors encoding my gene of interest under CMV promoter. The reporter gene is GFP driven by SFFV. While I get good transduction and visible GFP in HEK cells, when I take it to DRG cultures I can't pick up the fluorescence, even after 5days and using GFP antibodies. I suspect that the SFFV promoter just doesn't work very well in DRG.. Any experiences/thoughts?

PS: We have used constructs where GFP is driven by CMV (+WPRE) in DRG cultures before, with great results..

Thanks!

[gfischer]

biobio, on Mar 24 2010, 11:36 AM, said:

Hello all,

I have made some lentiviral vectors encoding my gene of interest under CMV promoter. The reporter gene is GFP driven by SFFV. While I get good transduction and visible GFP in HEK cells, when I take it to DRG cultures I can't pick up the fluorescence, even after 5days and using GFP antibodies. I suspect that the SFFV promoter just doesn't work very well in DRG.. Any experiences/thoughts?

PS: We have used constructs where GFP is driven by CMV (+WPRE) in DRG cultures before, with great results..

Thanks!

You could do PCR of gDNA to check for integration of the transgene cassette, but I suspect that the problem is that the promoter doesn't work in DRG.  The SFFV promoter seems to be used mostly in blood cells, may I ask why you're looking at this promoter?  If you're looking for neuron specific expression, neuron-specific enolase and synapsin are good choices.  For glial expression, GFAP is a good choice.

[biobio]

gfischer, on Mar 24 2010, 12:34 PM, said:

biobio, on Mar 24 2010, 11:36 AM, said:

Hello all,

I have made some lentiviral vectors encoding my gene of interest under CMV promoter. The reporter gene is GFP driven by SFFV. While I get good transduction and visible GFP in HEK cells, when I take it to DRG cultures I can't pick up the fluorescence, even after 5days and using GFP antibodies. I suspect that the SFFV promoter just doesn't work very well in DRG.. Any experiences/thoughts?

PS: We have used constructs where GFP is driven by CMV (+WPRE) in DRG cultures before, with great results..

Thanks!

You could do PCR of gDNA to check for integration of the transgene cassette, but I suspect that the problem is that the promoter doesn't work in DRG.  The SFFV promoter seems to be used mostly in blood cells, may I ask why you're looking at this promoter?  If you're looking for neuron specific expression, neuron-specific enolase and synapsin are good choices.  For glial expression, GFAP is a good choice.

Hello gfischer, thanks for your reply..

The only reason I used SFFV was that the cassette was available from a colleague, who has used it succesfully in other neuronal cells (cortical neurons if im not mistaken). I have arrived at the end of my PhD so making a new construct is not an option. A bit of a shame really.

I will still try to detect the overexpressed gene by qPCR in DRG, to make sure it's the SFFV-GFP that is messing up.

Thanks for the tips - will maybe use them in my post doc!

By the way these are non-integrating vectors, but it should still work the same (the transgenes are retained in episomal form).