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Abstract for Advances in Luciferase Reporter Technologies
Brad Swanson, Aileen Paguio, Denise Garvin, Pete Stecha, Frank
Fan and Keith Wood
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
Luciferase reporter assays are critical tools for the cancer
researcher because they are well
suited for quantifying important cellular processes including
proliferation, differentiation,
and death. This is due to their wide dynamic range, low endogenous
activity, and ease of
use. In particular, luciferase reporter assays have been crucial
tools in elucidating
transcriptional regulatory mechanisms that control cell many aspects
of cell physiology and
function. Recent improvements within the pGL4 Family of Luciferase Reporter
Vectors have
further improved the utility of these vectors to meet the demands
researchers in both
academic and pharmaceutical settings. Our efforts to improve the luciferase reporter gene
have enhanced both the dynamic range and the temporal response of
luciferase reporter
assays. We codon optimized the luc2 and hRluc reporter gene open
reading frames to
facilitate more efficient expression leading to higher levels of
luciferase protein.
Comparison of several different cell lines transfected with luc2
versus luc+ reporter vectors
validates that luc2 transfected cells have significantly higher
light output than luc+
transfected cells. To enable more rapid assay kinetics and reducing
assay time, we
incorporated protein destabilization domains into the luciferase
protein sequence. The
combination of these improvements results in a reporter gene that
allows high luciferase
protein expression that closely mirrors the transcriptional status
of the luciferase gene. In
addition the pGL4 Vector backbone has been optimized to
significantly reduce the number
of potential consensus transcription factor binding sites and lower
uninduced levels of the
luciferase reporter. These improvements have been incorporated into
our GloResponse™
Reporter Cell Lines to generate high quality stable reporter cell
lines that facilitate study of
the cAMP and NFAT signaling pathways in HEK293 cells. |
