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IRF3 knockout dual reporter monocytes

Nucleic acid sensing signaling in THP1-Dual™ KO-IRF3 cells

THP1-Dual™ KO-IRF3 cells were generated from the THP1-Dual™ cell line, which is derived from the human THP-1 monocytes, through the stable knockout of the IRF3 gene. IRF3 (interferon regulatory factor 3) is a critical player in the innate immune responses to virus infections. Viral nucleic acids in the cytosol are recognized by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), or cytosolic DNA sensors (e.g. the cyclic GMP-AMP synthase cGAS). Upon ligand binding, these receptors trigger the production of type I interferons (IFNs) through the activation of the TBK1/IKKε-IRF3/IRF7 pathway [1]. Activated PRRs also trigger the production of pro-inflammatory cytokines through activation of the NF-κB pathway [1].

THP1-Dual™ KO-IRF3 and THP1-Dual™ cells feature two reporter genes allowing the simultaneous study of the IRF pathway, by monitoring the activity of an inducible secreted Lucia luciferase, and the NF-κB pathway by monitoring the activity of an inducible SEAP (secreted embryonic alkaline phosphatase). Lucia luciferase and SEAP activities are readily assessable in the supernatant using QUANTI-Luc™ 4 Lucia/Gaussia and QUANTI-Blue™ Solution detection reagents, respectively.

As expected, the responses of THP1-Dual™ KO-IRF3 cells to the cyclic dinucleotide 2'3'-cGAMP (a STING agonist) and to lipopolysaccharide (a TLR4 agonist) are impaired. However, differential responses (either no effect, decrease or increase) are observed when using RNA or DNA agonists coupled with transfection reagents. THP1-Dual™ KO-IRF3 cells retain the ability to respond to cytokines such as type I IFNs and TNF-α. These cells are resistant to Blasticidin and Zeocin™.

Features of THP1-Dual™ KO-IRF3 cells:

Biallelic knockout of the IRF3 gene
Readily assessable Lucia luciferase and SEAP reporter activity

Applications for THP1-Dual™ KO-IRF3 cells:

Defining the role of IRF3 in PRR-induced signaling
Highlighting possible overlapping PRR activation or regulatory mechanisms (see 'Details' tab)

Validation of THP1-Dual™ KO-IRF3 cells:

IRF3 knockout verified by PCR, DNA sequencing, and western-blot
Functionally tested

References:

1. Iurescia S. et al., 2018. Nucleic acid sensing machinery: targeting innate immune system for cancer therapy. Recent Pat. Anticancer Drug Discov. 13: 2-17

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