Microscopic Analysis of Adenoviral Decontamination Using GFP Adenovirus with Comparable Sensitivity to Flow Cytometry

Abstract

Expression of transgenes from adenovirus vectors has become an extremely important and widely used tool in experimental cancer research and many other areas in the life sciences. It needs to be kept in mind, however, that adenoviruses are human pathogens and avoiding exposure of laboratory personnel to infectious viral particles is therefore an important concern. This issue seems even more important when the transgenes expressed for experimental purposes include oncogenic sequences. Decontamination procedures are thus required, whenever laboratory experiments with adenovirus vectors are performed and the effectiveness of these procedures has to be established. While many reports exist on the decontamination of blood and pharmaceutical products, data on the stability of adenoviruses during experiments performed in most life science laboratories are very scarce. One reason for this is that many of the methods used for assessing viral decontamination are time consuming and laborious and cannot easily be incorporated into the broad range of experimental setups typically performed in the laboratory. In this chapter we describe a reliable, sensitive, and simple method for the assessment of adenovirus decontamination by the use of an adenovirus expressing green fluorescent protein (GFP). The GFP adenovirus is subjected to various test conditions and afterwards susceptible indicator cells are exposed to the recovered virions. GFP expression is detected by a combination of fluorescence microscopy and flow cytometry. The simplicity and flexibility of the method allows one to monitor viral decontamination during the different scenarios occurring in the life science laboratory.

Canine and human sarcomas exhibit predominant FGFR1 expression and impaired viability after inhibition of signaling

“…Constant numbers of cells were incubated with varying numbers of virus termed multiplicity of infection (MOIs). As virus uptake control, an AV expressing enhanced green fluorescent protein (GFP) was used . Expression ratio of recombinant exogenous dnFGFR1 to endogenous FGFR1 was measured with primer assays detecting GFP and the kinase domain from canine and human FGFR1, respectively (suppl.…”