Q. Will the adenovirus work with my specific cells or tissue?
A. The Adenovirus has a very broad host range; it can infect human
and other mammalian cell lines or primary cells, including replicative
as well as non-replicative cells. There are in fact very few cell lines
that cannot be infected. Some lymphoid cell lines may be more resistant
to Adenovirus infection, and so may need high quantities of viruses to
achieve sufficient infection levels.
In general, the dose needed to infect 100% of cells varies considerably
from one cell type or tissue to another. We strongly recommend performing
an infectivity test with a control reporter virus (i.e. Ad5.CMV-GFP or
Ad5.CMV-LacZ) when starting a new project with recombinant Adenoviruses.
Q. What determines the tropism of Adenovirus in infecting cells?
A. Cell lines with a high number of Adenovirus receptors (CAR
and Integrin), like Cos-7 and HeLa, typically show significant Adenovirus
infection. Cell lines with lower numbers of adenovirus receptors, like
NIH 3T3 and U-937, exhibit lower levels of infection and need higher doses
(MOI) of viruses to infect all the cells.
Seth, P., M. Rosenfeld, et al. (1994). "Mechanism of enhancement
of DNA expression consequent to cointernalization of a replication-deficient
adenovirus and unmodified plasmid DNA." J Virol68(2):
Q. What MOI (multiplicity of infection) should I use with my cells?
A. An MOI (number of virus per cell) of 10 is suitable to infect
293 cells. An MOI range between 1 and 200 should be tested if susceptibility
to Adenovirus infection is unknown. The MOI may be increased up to 1000
when testing lymphoid cell lines. As a general guideline, the transfer
of a reporter gene to 100% of cells, without any signs of toxicity, can
generally be achieved with an MOI of 10-100 for most cell lines.
Q. Are the AdenoExpress adenoviruses safe to handle?
A. Except for the wild type AdenoExpressTM, the recombinant adenoviruses
we supply are defective viruses that are deleted in the E1 and E3 regions;
they will not replicate in cells other than complementing cells (293 cells).
According to references issued by the NIH (National Institute of Health)
Office of Biosafety, U.S. Department of Health, all serotypes of human
adenoviruses have been classified in biosafety level II. Level II consists
of agents that are to be considered of ordinary potential harm.
For more information on biosafety levels, please refer to the following
CDC publication: Biosafety in Microbiological and Biomedical Laboratories,
4th Edition, May 1999; this publication is also available at http://bmbl.od.nih.gov
Q. If I have a virus expressing the GFP (Ad5.CMV-GFP or Ad5.CMV5-GFP),
how soon after infection can I start seeing some fluorescence?
A. Infected cells should start expressing a detectable level of
GFP or BFP 4-20 hours after infection. The level of expression will vary
with the promoter used and the cell type so in some instances it may take
up to 24 hours. The GFP, being a stable protein builds up in the cell
whereas an unstable protein will be degraded in the cell. Accordingly,
the concentration of the GFP will increase over time.