6 replies to this topic

[nastyferatu]

Hi all,

I have a vector encoding GFP under UbC promoter activity. It's arrested by a floxed STOP signal (SV40).

• When I used it to transfect cells in vitro, N2A with FuGENE, in combination with a CRE vector, CRE recombined the STOP and GFP was expressed AND detected by fluorescent microscopy.

• HOWEVER, the same vectors when electroporated in utero in living tissue don't work properly: GFP is expressed, because I can detect it by immunostaining BUT GFP IS NOT FLUORESCENT, since I cannot find it by confocal microscopy.

Anyone can help me with this? How is it possible??

THANKS FOR YOUR TIME!

[gfischer]

How are you preparing the tissue?

Some methods of tissue processing can quench the fluorescence of GFP.

[bob1]

Perhaps the IF is more sensitive than the actual GFP - i.e. the GFP is below the detection limits for your imaging system.

It could also be that only part of the protein is being produced and the IF antibody is picking this up.

[nastyferatu]

To gfischer: I'm fixing the tissue with PFA 4%, by immersion of the brain after dissection. A normal and very well known method. It must work with this fixative. Moreover, it's the same fixative I'm using for transfected cells and they show GFP fluorescence.

To bob1: I think your first explanation could be right. I'm maxipreping the vector to electroporate more DNA. Moreover, I'm changing t
he promoter of the vector (replacing UbC by CAG). Regarding your second argument, I don't think a partial production is the reason because the protein and its fluorescence are fine in vitro.

THANKS FOR YOUR HELP!!

[leelee]

This may be a stupid suggestion, but do you have a GFP +ve control which you can look at with the confocal? Just to make sure you've got the settings and focus right etc?

I only suggest this because one of our students was having trouble picking up some GFP virus and it turned out she was just a little out of focus, making the GFP very difficult for her to find. This was with a fluorescent microscope.....

[nastyferatu]

Yes, I have a positive control and I easily find GFP at the confocal on it.

Thanks!

[CloneMan]

It could be that transfection efficiency is really low for the tissue, and you need both Cre and UBC-LoxP-STOP-LoxP-GFP plasmids to be transfected into the tissue to see GFP expression. Its certainly harder to electroporate 2 plasmids into the tissues than only 1 plasmid (for your control GFP).

We used to use Cre plasmid for transfection cells, but we switched to use Cre adenovirus for cell infection instead.