pNiFty2-SEAP

NF-kB-inducible SEAP reporter plasmid - ZeocinR

pNiFty2-SEAP plasmid is composed of three key elements: an ELAM proximal promoter, five NF-κB repeated transcription factor binding sites (TFBS) and a SEAP (Secreted alkaline phosphatase) reporter gene.

pNiFty2-SEAP plasmid is selectable with Zeocin® in both E. coli and mammalian cells, and can be used to generate stable clones.

Specifications

Transcription factor binding sites: NF-κB (5x)

Minimal Promoter: ELAM

Selection: Zeocin®

Reporter Gene: SEAP

Contents

• 20 µg of lyophilized DNA
• 1 ml of Zeocin® (100 mg/ml)

Product is shipped at room temperature.

Description

Minimal promoter

The proximal promoters are shorter than 500 bp and contain transcription factor binding sites. Upon stimulation in 293 cells, their expression level remains undetectable. With the addition of repeated TFBS, the proximal promoters become inducible by the appropriate stimulus and drive the expression of the reporter gene.

ELAM promoter: the proximal promoter of the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) gene contains three NF-κB sites and is truly NF-kB specific, as it lacks an AP1/CREB site found in the full-length promoter [1, 2].

Transcription factor binding sites (TFBS)

NF-kB binding site: Nuclear factor (NF)-κB is a “rapid-acting” primary transcription factor activated by a wide variety of PRRs. NF-κB is a protein complex that belongs to the Rel-homology domain-containing protein family. The prototypical NF-κB is composed of the p65(RelA) and p50 subunits [3]. NF-κB binds specific decameric DNA sequences (GGGRNNYYCC, R-purine Y=pyrimidine) and activates genes involved in the regulation of the innate and adaptative immune response.

Reporter Genes

SEAP reporter gene: Secreted alkaline phosphatase (SEAP) is a reporter widely used to study promoter activity or gene expression. SEAP expression can be rapidly and readily measured in supernatants of transfected cells. SEAP levels can be evaluated qualitatively with the naked eye and quantitatively using SEAP detection media, such as HEK-Blue™ Detection system.

References:

1. Schindler U., Baichwal VR., 1994. Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol. 14(9):5820-31.
2. Jensen LE. & Whitehead AS., 2003. ELAM-1/E-selectin promoter contains an inducible AP-1/CREB site and is not NFkB-specific. Biotechniques 35:54-58.
3. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med. 13(11):460-9.

Details