I would like to determine the effect of the overexpression of a certain protein on proliferation. I read in a publication that they delivered the gene with an AdV, induced expression and incubated for 3 weeks (followed by staining of the cells and determining the covered area of control and overexpression).
I was wondering if there are no problems resulting from the loss of episomal AdV plasmid. The progeny cells wouldn't be overexpressing the protein anymore, right?
Indeed adenoviruses do not integrate (no more than any other piece of DNA, anyway). In our experience, expression of Ad-transduced proteins in rapidly proliferating cells is nearly entirely lost within one week. This is mainly due to progressive dilution of the virus, which does not replicate, into an increasing number of cells.
If it is compatible with your experimental goals, you can do repeated infections, but if I understand your question correctly it is probably not.
I'd concur on the suggestion to use retro- or lentiviruses.
Hi, I think to see the effect of over expression after such a long time one must do stable expression of the gene of interest. in transient expression it is not possible to look at the effect after such long time. In case of AdV/Lentivirus mediated gene delivery, the viral gene easily get integrated to the host genome so its is probably possible in that case.
Lentivirus will indeed get integrated in the cell genome, however, that is not the case for adenovirus mediated gene delivery.
Indeed adenoviruses do not integrate (no more than any other piece of DNA, anyway). In our experience, expression of Ad-transduced proteins in rapidly proliferating cells is nearly entirely lost within one week. This is mainly due to progressive dilution of the virus, which does not replicate, into an increasing number of cells.
If it is compatible with your experimental goals, you can do repeated infections, but if I understand your question correctly it is probably not.
I'd concur on the suggestion to use retro- or lentiviruses.
We measure the effect of our protein of interest on long-term proliferation using primary murine bone marrow. they grow for, say 5-6 (1:5)- passages ex vivo. We measure by FACS how many infected cells there are (GFP) so we can use the non-infected cells as internal control. When we express our protein,GFP-expressing cells take over the entire population, GFP alone will dissappear with the other cells. We use a retroviral vector, pMIGR-1 (Pear et al, 1998,Blood 92, p3780-92). Also we do ecotropic packaging, which is nice, as only mouse cells will be infected. Important point if you are looking at possible oncoproteins. If you are interested in the system, drop me a note and we can discuss things in more detail!
Hi,since AdV plasmid doesnt integrated in the genom,the possibility of episomal loss of the plasmid exist.thus cells wouldn't be able to overexpressed the interest protein.
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