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pNiFty3 - mIFN-β promoter - NFAT - ZeocinR - SEAP
pNiFty3-T-SEAP plasmid is composed of three key elements: the mouse interferon beta minimal promoter, five NFAT transcription factor binding sites and a SEAP (Secreted alkaline phosphatase) reporter gene.
pNiFty3-T-SEAP plasmid is selectable with Zeocin™ in both E. coli and mammalian cells, and can be used to generate stable clonesBack to the top
Transcription factor binding sites: NFAT (4x)
Minimal Promoter: mouse IFNβ promoter
Reporter Gene: SEAP
pNiFty3-T-SEAP plasmid is provided as 20 µg lyophilized DNA with 4 pouches of Fast-Media® (2 TB and 2 Agar), containing Zeocin™.
Products are shipped at room temperature and should be stored at -20°C.Back to the top
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 .
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 . 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.
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 or the SEAP Reporter Assay Kit.
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.
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