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Please save me, have trouble to make adenovirus from 293A cells


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#1 chensandro

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Posted 25 June 2010 - 11:01 AM

Hi, Guys:

I am making adenovirus for some mcherry tagged protein X. I have them cloned in pshuttle-CMV vectors and recombination in BJXXXX Bac cells. I also got one positive control from addgene: pAD-dsRed (should be ready for transfection). I compared my constructs with pAD-dsRed with PacI digestion. They look great. I collect enough PacI digested DNA for transfection of 293A cells (from invitrogen).

The problem is I don't have any mcherry or dsred detection after lipofectamine 2000 transfection (48 hours after). Here are my control:
1. psicoR, I got great GFP after 48hours. 2. pcDNA3-mcherry-X: Good mcherry expression. 3. pAD-mcherry-X and pAD-dsRed have nothing.

I believe I should be able to detect mcherry if they are making virus.

I also tried some old 293Ad cells from other lab. No lucky either.

Any suggestion?

Thank you so much

#2 NEI_Girl668

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Posted 16 July 2011 - 02:03 PM

There are two reasons:
1. Your promoter is NOT right and cannot drive gene expression in your cells.
2. You failed to clone mCherry into shuttle vector.




Hi, Guys:

I am making adenovirus for some mcherry tagged protein X. I have them cloned in pshuttle-CMV vectors and recombination in BJXXXX Bac cells. I also got one positive control from addgene: pAD-dsRed (should be ready for transfection). I compared my constructs with pAD-dsRed with PacI digestion. They look great. I collect enough PacI digested DNA for transfection of 293A cells (from invitrogen).

The problem is I don't have any mcherry or dsred detection after lipofectamine 2000 transfection (48 hours after). Here are my control:
1. psicoR, I got great GFP after 48hours. 2. pcDNA3-mcherry-X: Good mcherry expression. 3. pAD-mcherry-X and pAD-dsRed have nothing.

I believe I should be able to detect mcherry if they are making virus.

I also tried some old 293Ad cells from other lab. No lucky either.

Any suggestion?

Thank you so much






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