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Key words: adenoviral vector • viral transduction • EGFP • IN Cell Analyzer • gene delivery
Biological assays developed in cultured and primary cells can greatly aid secondary screening as well as lead and target validation, but their development presents several challenges. Establishing stable cell systems to express target genes of interest at detectable levels is time-consuming. In addition, cellular signaling is complex, with data often difficult to interpret reproducibly.
Engineered adenoviral vectors mitigate these challenges by providing facile ways to deliver key genes into target cells. The Ad-A-Gene adenoviral vector gene delivery system rapidly delivers a range of signal pathway “sensors” into mammalian target cells via viral transduction (1, 2). These sensors comprise a panel of cellular genes, each of which is fused as a chimera to either enhanced green fluorescent protein (EGFP) or emerald fluorescent protein (eFP), or a response element controlling the expression of the E. coli B nitroreductase (NTR) reporter gene (3–7). The chimeric genes become transiently expressed within transduced target cells, allowing for the development of cellular assays that exploit the signal readouts from the sensor gene markers.
This application note describes the performance of an EGFP reporter assay for assessing the translocation of mitogen-activated protein kinase-activated protein kinase2 (MAPKAP-k2). MAPK signaling pathways are instrumental in the transduction of extracellular signals into intracellular responses. For instance, one specific set of pathways mediated by p38MAPK, are activated in response to stresses such as heat shock, ultraviolet light, bacterial lipopolysaccharide (LPS) and to pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFa) and interleukin 6 (IL-6). MAPKAP-k2, a direct substrate of p38MAPK
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