NF-κB and ISG Dual Reporter RAW 264.7 Cells

Dual IRF-Lucia and MIP-2-SEAP mouse macrophage reporter cell line

RAW-Dual™ (IRF-Lucia/KI-[MIP-2]SEAP) cells were generated from RAW 264.7 mouse macrophages, which express many pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) TLR2 and TLR4 [1], the cytosolic DNA sensor (CDS) cGAS [2], and the cyclic dinucleotide (CDN) sensor STING [2].

RAW‑Dual™ cells stably express two reporter genes encoding SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase.

SEAP expression depends on the activation of the endogenous MIP-2 promoter. MIP‑2, also known as CXCL2, is the murine homolog of human IL-8, a chemokine produced in an NF-κB-dependent manner [3]. The MIP-2 ORF has been replaced by the SEAP ORF using knockin technology. Hence SEAP expression reports activation of NF-κB.

The Lucia luciferase gene is under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements (ISRE). It reports the activation of interferon regulatory factors (IRFs).

Both reporter proteins are secreted and readily measurable in the cell culture supernatant using  QUANTI-Blue Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent. Alternatively, SEAP activity can be detected using HEK-Blue™ Detection, a cell culture medium allowing real-time detection of SEAP.

As a result, RAW-Dual™ cells allow for simultaneous study of the NF-κB and IRF pathway by assessing the activity of SEAP and Lucia luciferase, respectively.

References:

1. Underhill DM. et al., 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 401:811-5.
2. Hornef MW. et al., 2003. Intracellular Recognition of Lipopolysaccharide by Toll-like Receptor 4 in Intestinal Epithelial Cells. J Exp Med.198:1225-35.
3. Tanaka Y. & Chen ZJ., 2012. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal. 5(214):ra20.
4. Kim DS. et al., 2003. NF-kappaB and c-Jundependent regulation of macrophage inflammatory protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells. Mol Immunol. 40(9):633-43.