help: anenovirus transfection - (Mar/13/2007 )

Hi everybody,

I am trying to express a GFP fusion protein in endothelial cells through adenovirus transfection. The expression system I used is Virapower from Invitrogen. MOI is around 100. I checked GFP by flouresence microsope. However I never got GFP expression in endothelial cells or 293 cells. While if I transfected the adenovirus plasmid vector into 293 cells by lipofectmin2000, many cells are GFP positive. Now I really don't know what happened to the advenovirus. Any input is appreciated.

bio

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How have you determined/defined MOI?
Not an expert with adenovirus transfection, but normally you would determine your titer by serial dilution on cells... (Bear in mind that the same virus stock can have different MOI's on different cells also).

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I determined pfu by OD260 after adenovirus was purified. Even I infected 293 cells at MOI of 1, majority of cells showed cytopathic effect within 1-2 days. Therefore, I think the adenovirus was transfected into the cells. However, I did not observe GFP expression at time point. I am not sure whether adenovirus can go through recombination and lose the GFP.

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Check if the promoter you are using to drive GFP expression is functional in the infected cells.

How long did you wait to check for GFP fluorescence? Try waiting for an extra 24 hr.

Even 1 Freeze thaw cycle of the virus can destroy it. So take care about that.

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I used CMV promoter to drive GFP expression. GFP can be expressed if I directly transfected the adenovirus plasmid DNA into cells by lipofectmin 2000.


I checked GFP expression 24 or 48 hrs after transfection. I will wait for antoher 48 hrs to see what will happen.


Thanks for your answer

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