pNiFty3-IAN-Lucia

pNiFty3-IAN-Lucia plasmid is composed of three key elements: the mouse interferon beta minimal promoter, repeated transcription factor binding sites: ISRE (5x) AP-1 (5x) NF-κB (5x) and a Lucia secreted luciferase (Lucia) reporter gene.

pNiFty3-IAN-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: ISRE (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)

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  [2].
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)  [3]. 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   [4].    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. Wesoly J. et al., 2007. STAT activation and  differential complex formation dictate selectivity of interferon  responses. Acta Biochim Pol. 54(1):27-38.
3. Hess J, et al., 2004. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 117(Pt 25):5965-73.
4. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med. 13(11):460-9.

Details