The pIκB-EGFP Vector encodes the IκB-EGFP Signaling Probe, which is a fusion of enhanced green
fluorescent protein (EGFP) and IκB. IκB is an inhibitor of NFκB, a transcription factor involved in the
immune response and inflammatory diseases. When the NFκB pathway is inactive, IκB and NFκB
exist as an inactive complex in the cytosol. Upon stimulation, IκB is degraded. In cells transfected
with pIκB-EGFP, degradation of the IκB-EGFP fusion protein is observed as a decrease in EGFP
fluorescence (1). The IκB-EGFP Signaling Probe is constitutively expressed and resides in the
cytosol.
EGFP is a red-shifted, human codon-optimized variant of GFP (2–6) that has been engineered for
brighter fluorescence and higher expression in mammalian cells. Its excitation maximum is 488 nm
and emission maximum is 509 nm. For more information on the properties of EGFP, please refer to
the BD Living Colors? User Manual (PT2040-1) included with the vector.
Expression of the IκB-EGFP is driven by the human CMV immediate-early promoter. The SV40 poly-A
sequence directs proper processing of the 3' end of the fusion construct. The vector backbone also
contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A
neomycin resistance cassette (Neor) allows kanamycin selection in E. coli and neomycin (G418)
selection in eukaryotic cells. This cassette consists of the SV40 early promoter, the neomycin/
kanamycin resistance gene of Tn5, and poly-A signals from the Herpes simplex virus thymidine
kinase (HSV TK) gene. The vector backbone also provides a pUC origin of replication for
propagation in E. coli and an f1 origin for single-stranded DNA production.
