Key words: adenoviral vector • viral transduction • EGFP • IN Cell Analyzer • gene delivery
There is a requirement in the drug discovery process to investigate lead candidate drugs arising from primary screens and to validate those compounds in suitable biological assays. There is also a need in early-stage drug discovery to identify and measure, as well as modulate the activity of, a plethora of molecular targets involved in signal transduction pathways. Use of biological assays in cultured and primary cells can greatly aid secondary screening and early-stage drug discovery. However, establishing stable cell systems that express target genes of interest at detectable levels can present a genuine challenge. As a way forward, the use of adenoviral vectors to deliver genes and generate transient expression of key gene targets in cells provides an alternative method to techniques such as plasmid transfection.
The Ad-A-Gene adenoviral vector gene delivery system is composed of a panel of genes, each of which is fused to either enhanced green fluorescent protein (EGFP) or Emerald fluorescent protein (eFP), or a response element controlling the expression of the nitroreductase (NTR) reporter gene. Each sensor gene can be rapidly delivered by viral transduction of target cells, resulting in transient gene expression. Signal readouts from the sensor gene markers may then be exploited in the facile development of cellular assays that can report the status of a range of molecular targets at high levels of sensitivity.
This application note presents data from an EGFP translocation assay focusing on the glucocorticoid receptor (GCCR), which is a member of the steroid receptor superfamily. GCCR resides predominantly in the cytoplasm and when glucocorticoids bind to a defined ligand-binding site, the ligand-occupied GCCR translocates to the nucleus.
Translocation of GCCR w