Will the adenovirus work with my specific cells or tissue?

What determines the tropism of Adenovirus in infecting cells?

What MOI (multiplicity of infection) should I use with my cells?

Are the AdenoExpress adenoviruses safe to handle?

Do I risk losing titer after freezing and thawing my viral preparation?

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?

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, replicative as well as non-replicative cells. There are in fact, very few cell lines that will not be infected. Some lymphoid cell lines might be more resistant to Ad infection, but will 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, while cell lines with low numbers of adenovirus receptors (e.g. NIH 3T3 and U-937) show little and need higher doses (MOI) of viruses to infect all the cells.

Reference: 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 Virol 68(2): 933-40..

Q. What MOI (multiplicity of infection) should I use with my cells?

A. A MOI of 10 is suitable to infect 293 cells. A MOI (number of virus per cell) 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 a MOI of 10-100 for most cell lines.

Q. Are the AdenoExpress™ adenoviruses safe to handle?

A. Except for the wild type AdenoExpress™, 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. Do I risk losing titer after freezing and thawing my viral preparation?

A. The AdenoExpress™ viruses are CsCl purified and the buffer's composition and pH, which are critical for preservation of the titer upon freezing the particles, are optimized in our preparations of AdenoExpress™ viruses so that practically no loss of titer can be observed after freezing and thawing the viruses.

For optimal stability upon storage, we use the following buffer: Tris 10mM, pH 8.0, with 2 mM MgCl2 and 4% sucrose (Nyberg-Hoffman, C. and E. Aguilar-Cordova (1999). "Instability of adenoviral vectors during transport and its implication for clinical studies." Nat Med 5(8): 955-7.

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 8-20 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.