pNiFty3-AN-Lucia

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

pNiFty3-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: 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-κB, IRF3 and IRF7 [1].

Transcription factor binding sites (TFBS)

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) [2]. 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 [3].     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 luciferase 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. Hess J, et al., 2004. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 117(Pt 25):5965-73.
3. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med. 13(11):460-9.

Details