Figure 1

Schematic representation of the cloning-free reporter construction and assay and use in inflammation model. (A) An expression plasmid harboring a reporter cDNA is used as a template for PCR. Forward primer sequence contains 3' end that targets a minimal promoter region and 5'end that accommodates cis-acting sites. The reverse universal primer targets a region downstream of the polyA site. Purified PCR products are transfected into mammalian cells to express the reporter.(B) HEK293 cells in 96-well plates were transfected with 75 ng of purified PCR products in which the forward primer contains two copies of NF-κB site or a mutant form (Table 1, SEQ 1 and 2). After 20 hr, the cells were treated with IL-1α (log_0.5) for additional 20 hrs. (B, insert). Dose-response relationships between IL-1 and NF-κB mediated GFP reporter activity (normalized to basal levels of mutant NF-κB reporter levels). (C) EGFP activity was measured after 4 hr of IL-1 treatment with or without normalization (insert) with non-responsive RFP-PCR product. All data above are Mean ± SEM (quadruplicate) of one of three experiments. **p < 0.01, *** < 0.001. (D) Total RNA was extracted from HEK293 cells that were transfected with NF-κB responsive or mutant NF-κB-bearing PCR products in the absence or presence of IL-α (10 ng/ml) for 4 or 6 hrs. RT-PCR was performed using primers specific to EGFP as described in Methods. PCR products were run on gel, and β-actin normalized signal intensities of ethidium bromide-stained products were quantitated using AlphaEase software. Data are from three experiments.(E) Dose-dependent curve of TNF-α action on reporter activity produced from NF-κB responsive linear PCR construct.