TLR2 KO Dual Reporter THP-1 Cells

Notification: Reference #thpd-kotlr2-av can only be ordered together with reference #thpd-kotlr2.

TLR2 knockout dual reporter monocytes

NF-κB and IRF signaling pathways in THP1-Dual™ KO-TLR2 cells

InvivoGen offers a human monocyte-derived cell line specifically designed for the study of human TLR2 (Toll-like receptor 2) function:

• THP1-Dual™ KO-TLR2 cells

THP1-Dual™ KO-TLR2 cells were generated from the THP1-Dual™ cell line through the stable knockout of the TLR2 gene. They feature two inducible reporter genes, allowing the concomitant study of the IRF and NF-κB pathways, by monitoring the Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase) activities, respectively.
As expected, the NF-κB-mediated response is abolished in THP1-Dual™ KO-TLR2 cells upon incubation with TLR2-specific ligands such as Pam3CSK4 (TLR2/1), FSL-1 (TLR2/6), and heat-killed Listeria monocytogenes (HKLM; TLR2/6) when compared to the THP1-Dual™ cells, with no notable difference for the other ligands tested. Additionally, as TLR2 does not directly signal through an IRF-dependent pathway, the secretion of Lucia luciferase is unaltered in THP1-Dual™ KO-TLR2 when tested across a range of IRF-inducing ligands (see Figures).

Background:

Toll-like receptor 2 (TLR2) plays an essential role in detecting a diverse range of microbial pathogen-associated molecular patterns (PAMPs) from bacteria, fungi, and parasites, including lipoproteins, lipoteichoic acid, lipoarabinomannan, and chitin [1]. A number of viruses have also been shown to interact directly with TLR2, including HIV and herpes simplex virus [1, 2]. TLR2 forms a heterodimer on the cell surface with either of its co-receptors, TLR1 or TLR6, which is crucial for signaling and ligand specificity. The TLR2/TLR1 and TLR2/TLR6 heterodimers specifically bind lipoproteins depending on whether they are tri- or diacylated, respectively [1]. Their activation triggers NF-κB- and AP-1-mediated pro-inflammatory responses [3].

Learn more about TLR2 heterodimers.

Key Features:

• Verified knockout of the TLR2 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

Applications:

• Defining the role of TLR2 in PRR-induced signaling, or related cell signaling pathways 
• Exclusion of contaminating TLR2 agonist-dependent (e.g. bacterial lipoproteins) signaling
• Highlighting possible overlap between TLR2 and other signaling pathways

References

1. Oliveira-Nascimento, L. et al. 2012. The Role of TLR2 in Infection and Immunity. Front Immunol 3, 79.
2. Henrick, B.M. et al. 2015. HIV-1 Structural Proteins Serve as PAMPs for TLR2 Heterodimers Significantly Increasing Infection and Innate Immune Activation. Front Immunol 6, 426.
3. Li, J. et al. 2013. Evolving Bacterial Envelopes and Plasticity of TLR2-Dependent Responses: Basic Research and Translational Opportunities. Front Immunol 4, 347.

Figures

Validation of TLR2 KO: (A)The targeted TLR2 region in THP1-Dual™ (WT; blue arrow) parental cells and THP1-Dual™ KO-TLR2 (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-TLR2 cells feature a frameshift deletion, causing an early stop codon and inactivation of TLR2. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-TLR2 (KO) cells were analyzed using an anti-human TLR2 antibody (green arrow), followed by an HRP‑conjugated anti‑rabbit secondary antibody. As expected a band was detected at ~90 kDa in the WT cells only.

NF-κB responses in THP1-Dual™-derived cells: THP1-Dual™ and THP1-Dual™ KO-TLR2 cells were incubated with 0.3 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), 300 ng/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 1 μg/ml LPS-EK Ultrapure (UP; TLR4), 1 ng/ml Pam3CSK4 (TLR2/1 agonist), 0.3 ng/ml FSL-1 (TLR2/6 agonist), 107 c/ml HKLM (TLR2 agonist), and 3 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).

IRF responses in THP1-Dual™-derived cells: THP1-Dual™ and THP1-Dual™ KO-TLR2 cells were incubated with 0.3 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), 300 ng/ml 3php‑RNA/LyoVec™ (RIG-I agonist), 1 μg/ml LPS-EK Ultrapure (UP; TLR4), 1 ng/ml Pam3CSK4 (TLR2/1 agonist), 0.3 ng/ml FSL-1 (TLR2/6 agonist), 107 c/ml HKLM (TLR2 agonist), and 3 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).

 

Specifications

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™

Antibiotic resistance: Blasticidin and Zeocin®

Quality Control:

• Biallelic TLR2 knockout has been verified by PCR, DNA sequencing, and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ THP1-Dual™ KO-TLR2 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 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)