Two years ago I had tried to transfect Huh-7 cells with the same adenovirus and got good protein expression of the insert as well as GFP reporter gene.
I´m trying now to do the same with very bad results. I have no overexpression of my protein (western blot) nore good overexpression of GFP (fluorescense microscopy), Just 4-6 green cells/field (10x).
I tryed with viruses of the same batch of two years ago and with viruses of a new batch having the same negative results.
When I try both batches of viruses with isolated hepatocytes culture they have good tranfection and express GFP and my insert.
1) check the insert in vector: it may be lost or decreased within 2 years
2) calculate viability of cells after transfection, if apoptosis exceeds 10%, thaw another culture
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