pNiFty3-TAN-SEAP

pNiFty3-TAN-SEAP plasmid is composed of three key elements:  the mouse interferon beta minimal promoter,  repeated TFBS (transcription factor binding sites):  NFAT (5x) AP-1 (5x) NF-κB (5x) and a  SEAP (Secreted alkaline phosphatase) reporter gene.

pNiFty3-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: NFAT (5x) AP-1 (5x) NF-κB (5x)
Minimal Promoter: mouse IFNβ promoter
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.

IFN-β promoter: the mouse IFN-β minimal promoter comprises several positive regulatory domains that bind different cooperating transcription factors such as NF-kB, IRF3 and IRF7 [1].

Transcription factor binding sites (TFBS)

NFAT binding site: Nuclear factor of activated T-cell (NFAT) is a family of transcription factors expressed in T cells, but also in other classes of immune and non-immune cells [2]. NFAT is activated by stimulation of receptors coupled to calcium mobilization, such as the PRRs Dectin-1 and Mincle [3, 4]. Calcium mobilization induces the calmodulin-dependent phosphatase calcineurin leading to NFAT activation. NFAT binds to a 9 bp element, with the consensus sequence (A/T)GGAAA(A/N)(A/T/C)N.
AP-1 binding site: Activator protein 1 (AP-1) is a transcription factor activated by most PRRs. AP-1 is a  heterodimeric complex composed of members of Fos, Jun and, ATF protein families. AP-1 binds to the TPA responsive element (TRE: ; TGAG/CTCA) [5]. AP-1 activation in TLR signaling is mostly mediated by MAP kinases such as c-Jun N-terminal kinase (JNK), p38 and extracellular signal regulated kinase (ERK).
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 [6]. 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 Gene

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.

1. Vodjdani G. et al., 1988. Structure and characterization of a murine chromosomal fragment containing the interferon beta gene. J Mol Biol. 204(2):221-31.
2. Rao A. et al., 1997. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 15:707-47.
3. Goodridge HS. et al., 2007. Dectin-1 stimulation by Candida albicans yeast or zymosan triggers NFAT activation in macrophages and dendritic cells. J Immunol. 178(5):3107-15.
4. Yamasaki S. et al., 2009. C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia. PNAS. 106(6):1897-902.
5. Hess J, et al., 2004. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 117(Pt 25):5965-73.
6. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med. 13(11):460-9.

Details