[Histonet] expression pattern of GFP fusion protein in cells and mouse

[koellingr <@t> comcast.net]

DONGTAO FU, 



Was hoping to see some response for curiosity sake but haven't so this is my take.  In a former life have seen such an occurence. Not with these same reagents but similar happenings. And while I didn't take the time to investigate exactly why, we attributed it to posttranslational modifications. 



Those 293 cells are human kidney cells and while cells they are certainly different from mouse cells.  When you put in that AAV/dystrophin/GFP cassette into 293 or mice or any kind of other expression model, while they might be transcribed similarly, each cell type (species) has it's own unique way of posttranslational modifications to the protein. Acetylation, alkylation, glycosylation, and hundreds of others, any one of which could modify an epitope or cover it up.  There are articles innumerable on how posttranslational modifications, different in different species,  can affect  protein composition and shape (and obviously that could very well impact ability to see it with an antibody that might work perfectly well in detection in another system). 



Ray Koelling 

PhenoPtah Labs 

Seattle, WA 
----- Original Message ----- 
From: "DONGTAO FU" <fudo <@t> ufl.edu> 
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Thursday, February 19, 2009 6:48:58 AM GMT -08:00 US/Canada Pacific 
Subject: [Histonet] expression pattern of GFP fusion protein in cells and mouse 

Hi, all 

  A researcher met a problem of GFP fusion protein expression and 
is searching an answer from the specialists here. The following is 
his question: 

  I am working with an AAV vector which contains a transgene 
(dystrophin) which is fused to GFP. When I inject this vector in 
mice, and stain the tissues with antibodies raised against GFP and 
dystrophin, I only pick up GFP. It is blazing. I was surprised not 
to see colocalization given that this is a fusion protein. (I used 
the same immunostaining protocol that others have successfully 
used before me to pick up dystrophin). So, the next thing I did 
was infect 293 cells with the same vector. When I stained those 
cells, I picked up beautiful expression of dystrophin and GFP. 
They were perfectly colocalized, as expected. So, my question is, 
have you seen examples where certain epitopes are masked in one 
cellular environment and not in another? (maybe the reason that I 
cannot detect the dystrophin in the mouse tissues, but I can in 
293 cells). 

  Does anyone know the possible answer of this phenomenon? Any 
thoughts will be greatly appreciated. 

Ann 


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