pE2F-TA-Luc is designed to monitor the induction of E2F-mediated signal transduction pathways.
E2F, a major target of the retinoblastoma gene (Rb), is a key regulator of cell-cycle checkpoints
in mammalian cells (1–2). E2F plays a critical role in stimulating expression of genes encoding
growth-promoting proteins (3), and is involved in regulating the expression of important genes
during cell proliferation (4–5). The E2F protein forms a heterodimer complex with the DP1
protein, which binds to E2F response elements and initiates transcription of genes necessary
for DNA replication. Studies have shown that deregulation of E2F results in a loss of cell-cycle
checkpoints—thereby predisposing cells to uncontrolled growth (1–5). pE2F-TA-Luc contains
four copies of the E2F enhancer element (6), located upstream of the minimal TA promoter, the
TATA box from the herpes simplex virus thymidine kinase promoter (PTA). Located downstream
of PTA is the firefly luciferase reporter gene (luc). Upon binding of the E2F/DP1 complex to the
cis-acting E2F enhancer element, transcription is induced and the reporter gene is activated.
The luciferase coding sequence is followed by the SV40 late polyadenylation signal to ensure
proper, efficient processing of the luciferase transcript in eukaryotic cells. A synthetic transcription
blocker (TB) is located upstream of the cis-acting enhancer element. It is composed of
adjacent polyadenylation and transcription pause sites for blocking nonspecific transcription
(7). The vector backbone also contains an f1 origin for single-stranded DNA production, a pUC
origin of replication, and an ampicillin resistance gene for propagation and selection in E. coli.