IRF3 KO Dual Reporter THP1 Cells

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

Figures

Amplification of the targeted IRF3 region in THP1-Dual™ (WT) and THP1-Dual™ KO-IRF3 (KO) cells. THP1-Dual™ KO-IRF3 cells feature a biallelic deletion of 70 base pairs (arrow).

Analysis of lysates from the THP1-Dual™ (WT) and THP1-Dual™ KO-IRF3 (KO) using Anti-IRF3 (10 μg/ml), followed by HRP-conjugated anti-mouse secondary antibody (undiluted). The arrow indicates the expected band for the IRF3 protein (47 KDa).

2x10⁵ THP1-Dual™ (WT) and THP1-Dual™ KO-IRF3 (KO-IRF3) cells were incubated with 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, IRF (A) and NF-κB (B) responses were assessed by measuring the activity of Lucia luciferase and SEAP in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution, respectively. Activity fold increase over non-induced cells (Lucia luciferase readout) or reading of optical density (OD) at 630 nm (SEAP readout) are shown.

2x10⁵ THP1-Dual™ or THP1-Dual™ KO-IRF3 cells were transfected with 1 μg/ml 3p-hpRNA or 1 μg/ml 5’ppp-dsRNA complexed with Lyovec™ (A, B) or LTX (C, D). After overnight incubation, IRF response was assessed by measuring bioluminescent activity of the Lucia luciferase in the supernatant using QUANTI-Luc™. Activity fold increase over non-transfected cells is shown (A, C). The NF-κB activity in THP1-Dual™-derived cells was assessed by measuring the SEAP activity in the supernatant using QUANTIBlue™ Solution. Reading of optical density (OD) at 630 nm is shown (B, D).

2x10⁵ THP1-Dual™ or THP1-Dual™ KO-IRF3 cells were incubated with 10 ng/ml human TNF-α (hTFN-α), 104 U/ml human IFN-α (hIFN-α), 104 U/ml human IFN-β (hIFN-β), or 1 μg/ml LPS EK Ultrapure (UP). After overnight incubation, IRF (A) and NF-κB (B) responses were assessed by measuring the activity of Lucia luciferase and SEAP in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution, respectively. Activity fold increase over non-induced cells (Lucia luciferase readout) or reading of optical density (OD) at 630 nm (SEAP readout) are shown.

Specifications

Antibiotic resistance: Blasticidin and Zeocin™

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Quality Control:

• Biallelic IRF3 knockout has been verified by PCR, DNA sequencing, western blot, and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Contents

• 1 vial of THP1-Dual™ KO-IRF3 cells (3-7 x 10⁶ cells) in freezing medium
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of Zeocin™ (100 mg/ml)
• 1 ml of Blasticidin (10 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

 Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

THP1-Dual™ KO-IRF3 cells have been functionally tested using various sources of nucleic acids (see the validation data document).

As expected, IRF and NF-κB responses are severely impaired when the THP1-Dual™ KO-IRF3 cells are incubated with STING agonists, such as 2’3’-cGAMP, which do not require transfection to access the cytosol. However, differential responses are observed when using RNA agonists with transfection reagents. The weak agonist 5’ppp-dsRNA loses its ability to induce an IRF response in KO-IRF3 cells when complexed with LyoVec™ or LTX. On the contrary, the IRF response is unexpectedly increased when using the highly potent agonist 3p-hpRNA complexed to LyoVec™ or LTX. Surprisingly, with either agonist, the NF-κB response is barely affected. These data suggest that the use of different agonists and transfection reagents could highlight overlapping RNA-sensing or regulatory mechanisms.