TRIF KO Dual Reporter THP1 Cells

TRIF knockout dual reporter monocytes

THP1-Dual™ KO-TRIF cells were generated from the THP1-Dual™ cell line, which is derived from the human THP-1 monocytic cell line, through the stable knockout of the TRIF gene. THP1-Dual™ KO-TRIF 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™ and QUANTI-Blue™ Solution detection reagents, respectively.

TIR-domain-containing adapter-inducing interferon-β (TRIF), also known as TIR-containing adaptor molecule-1 (TICAM-1), is a key adaptor molecule in endosomal TLR-dependent signaling cascades. Upon recognition of lipopolysaccharide (LPS) by endocytosed TLR4, TRIF is responsible for mediating activation of interferon regulatory factor 3 (IRF3) and the production of type I interferons (e.g. IFNβ) [1]. Additionally, TRIF has been shown to induce TLR4-dependent 'late' activation of NF-κB. Importantly, the endosomal TLR4-TRIF signaling cascade is independent of the cell surface TLR4-MyD88 signaling pathway [2]. For comparing these two distinct TLR4 signaling cascades, InvivoGen also offers THP1-Dual™ KO-MyD cells.

NF-κB and IRF signaling pathways in THP1-Dual™ KO-TRIF cells

Key Features:

• Verified knockout of the TRIF gene (PCR, DNA sequencing, and functional assays)
• Functionally validated with a selection of PRR ligands and cytokines
• Readily assessable Lucia luciferase and SEAP reporter activities
• The stability for 20 passages, following thawing, has been verified
• Guaranteed mycoplasma-free

Applications:

• Defining the role of TRIF in PRR-induced signaling (i.e. TLR4), or related cell signaling pathways
• Highlighting possible overlap between TRIF and other signaling pathways

Please note: Studying the TRIF signaling pathway with THP1-Dual™-derived cells does not require PMA‑differentiation, however it does significantly increase their sensitivity to IRF-inducers (i.e. TLR4-dependent TRIF activation). Additionally, THP1-Dual™ cells do not respond to TLR3-specific ligands.

References:

1. Ullah, M.O. et al. 2016. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target. J Leukoc Biol 100, 27-45.
2. Ciesielska, A. et al. 2020. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci.

Figures

Validation of TRIF KO. (A) The targeted TRIF region in THP1-Dual™ (WT; blue arrow) parental cells and THP1‑Dual™ KO-TRIF (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-TRIF cells feature a frameshift deletion, causing an early stop codon and inactivation of TRIF. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-TRIF (KO) cells were analyzed using an anti-human TRIF antibody (green arrow), followed by an HRP‑conjugated anti‑rabbit secondary antibody. As expected a band was detected at ~100 kDa in the WT cells only.

IRF responses in PMA-differentiated THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TRIF cells were pretreated with PMA (Phorbol 12-myristate 13-acetate; 10 ng/ml for 3 hours). After 3 days recovery, the cells were incubated with 10 ng/ml human (h)TNF-α (NF‑κB‑SEAP positive control), 1 x 10⁴ U/ml hIFN-β (IRF-Lucia positive control), 1 μg/ml VACV70/LyoVec™ (CDS ligand), 1 µg/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 30 ng/ml LPS-EK Ultrapure (UP; TLR4), 30 ng/ml LPS-SM-UP (TLR4 agonist), 30 ng/ml CRX-527 (TLR4 agonist), 10 ng/ml Pam3CSK4 (TLR2/1 agonist), 10 µg/ml R848 (TLR7/8 agonist), and 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown as a fold increase over non-induced cells (Lucia luciferase readout).

NF-κB responses in PMA-differentiated THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TRIF cells were pretreated with PMA (Phorbol 12-myristate 13-acetate; 10 ng/ml for 3 hours). After 3 days recovery, the cells were incubated with 10 ng/ml human (h)TNF-α (NF‑κB‑SEAP positive control), 1 x 10⁴ U/ml hIFN-β (IRF-Lucia positive control), 1 μg/ml VACV70/LyoVec™ (CDS ligand), 1 µg/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 30 ng/ml LPS-EK Ultrapure (UP; TLR4), 30 ng/ml LPS-SM-UP (TLR4 agonist), 30 ng/ml CRX-527 (TLR4 agonist), 10 ng/ml Pam3CSK4 (TLR2/1 agonist), 10 µg/ml R848 (TLR7/8 agonist), and 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

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 TRIF 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. 

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 106 THP1-Dual™ KO-TRIF cells in a cryovial or shipping flask
• 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 pouch of QUANTI-Luc™
• 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 Shipped on dry ice (Europe, USA & Canada)

Details

TRIF Background

TIR-domain-containing adapter-inducing interferon-β (TRIF), also known as TIR-containing adaptor molecule-1 (TICAM-1), is a key adaptor molecule in TLR-dependent signaling cascades. Specifically, TRIF is responsible for mediating TLR4-dependent, MyD88 independent signaling upon recognition of lipopolysaccharide (LPS), as well as, TLR3-dependent signaling upon recognition of double-stranded (ds)RNA [1]. Ultimately, TRIF recruitment activates interferon regulatory factor-3 (IRF3) which regulates both type I interferon (IFN) gene expression and a range of IFN-stimulated genes (ISG) [1]. TRIF plays a central role in a range of innate immune responses including anti-viral host defenses, cell death, and homeostasis [1].

Additionally, TRIF has been shown to induce TLR4-dependent late activation of NF-κB through recruitment and activation of TRAF6 or receptor-interacting serine/threonine protein kinases 1 and 3 (RIPK1 and RIPK3) [2,3]. In addition to the key role played by RIPK1 & RIPK3 in necroptosis, their activation in this context has been shown to lead to the acute expression of pro-inflammatory cytokines [2]. Importantly, TLR4-dependent TRIF activation is independent of its activation of the MyD88-dependent (at the cell surface) pathway, which is implicated in ‘early’ activation of NF-κB and a subsequent pro-inflammatory response [3].

References:

1. Ullah, M.O. et al. 2016. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target. J Leukoc Biol 100, 27-45.
2. Najjar, M. et al. 2016. RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4. Immunity 45(1), 46-59.
3. Ciesielska, A. et al. 2020. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci.

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