pNiFty3-TAN-Lucia

pNiFty3-TAN-Lucia 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 secreted luciferase (Lucia) reporter gene.

pNiFty3-TAN-Lucia 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: Lucia luciferase

These products are covered by a Limited Use License (See Terms and Conditions).

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

Lucia luciferase reporter gene: Lucia is a secreted luciferase with strong bioluminescent activity
The     Lucia reporter gene is ideal for promoter activity and gene     expression  studies. Lucia is encoded by a synthetic gene derived by     genetic  engineering of copepod luciferase genes. Lucia luciferase     activity can  be detected and quantified directly in the culture medium     of  transfected cells using InvivoGen's QUANTI-Luc™ detection reagent.

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.

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