pGen.IRES.GFP.Neo - (Aug/03/2005 )

Hi,

I am currently using pGen.Ires.GFp to make stable cell lines. GFP is expressed from the IRES translation iniation sequence in this construct. However, after transfection, the cells do not fluoresce when viewed by fluorescent microscopy.
I know the transfection protocol is fine as control GFP plasmids( GFP expressed from a CMV promoter) give many bright fluorescent cells.
I was hoping to facs sort my cells as they express GFP - but Im not sure this will work now.
Anyone else used pGEN.IRES.GFP? Do you see fluorescent cells post transfection??

Thanks

Dear Paulaz,

I have not used the exact vector you mentioned, but have a lot of experience with other IRES-GFP plasmids. Firstly, when GFP is positioned behind the IRES, it will not "glow" as bright as it would directly behind a promoter.

Try visualising by flow cytometry - this is a very sensitive method and you should see a shift compared to untransfected cells, but it would not be as far right as a non-IRES GFP.

The second point is that the length of IRES can affect your GFP expression ... has this vector worked for other primary gene transfections??

Finally, if your transfection efficiency is low, combined with the decreased GFP expression level, you may not detect anything under the microscope. Again, use the flow cytometer.

Hope this helps,

AussieUSA.

Hi,

I´m also working with the vector you mentioned, and I tried to use the empty vector (without a gene cloned before IRES) for transfection. Now I can´t see any fluorescence, even in FACS. Does anyone know if EGFP expression is influenced if there´s no gene before IRES?

Thanks,
Susan

Which company did you get this vector? I want to find the map of this vector. Thanks