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Adenovirus production in 293 cells


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#1 aei8

aei8

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Posted 12 April 2009 - 06:28 AM

Hi there!

I'm trying to prepare an adenovirus using the Adeno1 expression system from Capital Biosciences. For this to work one clones the cDNA of interest into a shuttle vector, and then subclones a part of this shuttle vector into a larger plasmid thus creating the viral genome that includes the cDNA. Once this is done, the viral genome plasmid is cut with PacI, transfected into 293 cells, and then you wait for a cytopathic effect.

I've gone through all these stages and transfected 293 cells but got no cytopathic effect (at least not one that's visible) despite having waited for 3 weeks. I've used various lines of 293 cells for this, including 293A, and have repeated this several times - same result. The viral genome plasmid seems okay by restriction digest (plus I can see expression of my protein by Western blot a couple of days post-transfection), so I guess my viral plasmid is okay. The 293 cells I use are very capable of propagating existing adenoviruses - it's just the first step of moving from plasmid to virus that is messed up.

Does anybody know what's going on and how I can get a cytopathic effect?

Thanks!




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