Abstract
Virology has greatly benefited from the introduction of fluorescent proteins (FP's) as tags to viral as well as cellular structures. With advanced imaging technologies it is now possible to observe host-pathogen interactions in living cell systems in real-time. The generation of high-quality genetic tools to study host-pathogen interactions therefore becomes imperative for the further development of this type of analysis. In this chapter we describe a universal and reliable method to rapidly generate stable cell lines expressing FP-tagged proteins to be used for the analysis of host-pathogen interactions. The protocol is exemplified for two cellular structures recognized for their importance in the host-pathogen interplay: autophagosomes and the actin cytoskeleton, but can be applied to virtually any transgene or FP. It is based on the commercial Flp-In™ and Gateway™ systems (Life Technologies) and allows the rapid generation of FP-tagged transgenes by Gateway™ technology followed by recombination into a cell line containing a single transcriptionally active genomic recombination locus.
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