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- NF-kB binding site: Nuclear factor (NF)-kB is a “rapid-acting” primary
transcription factor activated by a wide variety of PRRs. NF-kB is a protein
complex that belongs to the Rel-homology domain-containing protein
family. The prototypical NF-kB is composed of the p65(RelA) and p50
subunits 7 . NF-kB binds specific decameric DNA sequences
(GGGRNNYYCC, R-purine Y=pyrimidine) and activates genes involved
in the regulation of the innate and adaptative immune response.
- ISRE binding site: PRRs involved in the antiviral response induce the
activation of interferon regulatory factors (IRFs) and the production of type
I interferons (IFNs). IFNs trigger the formation of the ISGF3 complex which
contains signal transducer and activator of transcription (STAT) 1, STAT2
and IRF9. ISGF3 and IRFs bind to specific nucleotide sequences called
interferon-stimulated response elements (ISREs; AGTTTCNNTTTCC) in
the promoter of IFN-stimulated genes (ISGs) leading to their activation 8 .
- 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 9 . NFAT is activated by stimulation of
receptors coupled to calcium mobilization, such as the PRRs Dectin-1
and Mincle 10, 11 . 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.
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, or the SEAP Reporter Assay Kit (see “Related Products”).
- Luc reporter gene: The firefly luciferase gene is a highly sensitive
reporter gene and thus is ideal for detecting low-level gene expression.
Luc activity can be quantified in cell extracts by using kits commercially
available from other companies.
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. 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. 4. Wathelet MG. et al., 1987. New inducers revealed
by the promoter sequence analysis of two interferon-activated human genes. Eur J
Biochem. 1987 Dec 1;169(2):313-21. 5. Grandvaux N, et al., 2002. Transcriptional
profiling of interferon regulatory factor 3 target genes: direct involvement in the
regulation of interferon-stimulated genes. J Virol. 2002 Jun;76(11):5532-9. 6. Hess J,
et al., 2004. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 117(Pt
25):5965-73. 7. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like
receptors. Trends Mol Med. 13(11):460-9. 8. Wesoly J. et al., 2007. STAT activation
and differential complex formation dictate selectivity of interferon responses. Acta
Biochim Pol. 54(1):27-38. 9. Rao A. et al., 1997. Transcription factors of the NFAT
family: regulation and function. Annu Rev Immunol. 15:707-47. 10. 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.
11. Yamasaki S. et al., 2009. C-type lectin Mincle is an activating receptor for
pathogenic fungus, Malassezia. PNAS. 106(6):1897-902.
Contents and Storage
pNiFty plasmids are provided as lyophilized transformed E. coli strains on
paper disk with 4 pouches of Fast-Media ® (2 TB and 2 Agar), containing
the appropriate antibiotic: ampicillin for pNiFty plasmids, or Zeocin for
pNiFty2 and pNiFty3 plasmids. Products are shipped at room
temperature and should be stored at -20ºC.
Related Products
www.invivogen.com/innate-immunity-pnifty
HEK-Blue Detection, page 15
QUANTI-Blue , page 14
SEAP Reporter Assay Kit, page 13
Fast-Media ® , page 45
Zeocin , page 18
AP-1
NF-kB
ISRE
NFAT
individually
or
combined
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INNATE IMMUNITY 3