TLR4 KO Reporter RAW 264.7 Cells

TLR4 knockout IRF-Lucia & MIP-2-knockin-SEAP murine macrophage reporter cell line

TLR and Type I IFN signaling in RAW-Dual™ KO-TLR4 Cells

RAW-Dual™ KO-TLR4 (IRF-Lucia/KI-[MIP-2]SEAP) cells were generated from RAW-Dual™ cells through stable gene knockout of Toll‑like receptor 4 (TLR4). These cells derive from RAW 264.7 murine macrophages which express many pattern recognition receptors (PRRs) [1].

RAW‑Dual™ KO-TLR4, along with their parental cell line RAW-Dual™ cells, can be used to:

• Investigate TLR4 signaling
• Study TLR4-independent biological activity of test compounds.

Both cell lines 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 is a chemokine produced in an NF-κB-dependent manner [2]. The MIP-2 ORF has been replaced by the SEAP ORF using knockin technology.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 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™ KO-TLR4 cells allow to simultaneously study the NF-κB pathway, by assessing the activity of SEAP, and the IRF pathway, by monitoring the activity of Lucia luciferase.

As expected, RAW-Dual™ KO-TLR4 cells do not respond to TLR4 agonists such as whole lipopolysaccharide (LPS) or monophosphoryl lipid A (MLPA).

RAW-Dual KO-TLR4™ cells are resistant to Zeocin®.

References:

1. West A. et al., 2011.TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472:476-80.
2. Kim D. et al., 2003. NF-kappaB and c-Jun-dependent regulation of macrophage inflammatory protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells. Mol Immunol. 40:633-43.

Figures

RAW-Dual™ KO‑TLR4 cells and RAW-Dual™ (parental cell line) cells were incubated with 1 μg/ml Pam3CSK4 (triacylated lipopeptide; TLR2 agonist), LPS‑EK Ultrapure (smooth LPS from E. coli K12; TLR4 agonist), LPSSM (rough LPS from S. minnesota R595; TLR4 agonist), MPLAs (synthetic monophosphoryl lipid A; TLR4 agonist), 10 μg/ml CL075 (thiazoloquinoline compound; TLR7 agonist), Poly(I:C)/LyoVec™ (dsRNA complexed with transfection reagent; RIG-I agonist) or MDP (muramyldipeptide; NOD1 agonist). After overnight incubation, NF‑κB activation was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm.

RAW-Dual™ KO‑TLR4 and RAW-Dual™ cells were stimulated with 1 x 10⁴ U/ml mIFN-β, 1 μg/ml LPS-EK Ultrapure, LPS‑SM Ultrapure, MPLAs, 10 μg/ml Poly(dA:dT)/LyoVec™ (dsDNA naked complexed with transfection reagent; CDS agonist), Poly(I:C)/LyoVec™ (RIG-I agonist) or 10 μg/ml 2’3’-cGAMP (cyclic dinucleotide; STING agonist). After overnight incubation, IRF activation was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI‑Luc™, a Lucia luciferase detection reagent. The IRF induction of each ligand is expressed relative to that of mIFN-β (taken as 100%).

Specifications

Antibiotic resistance: Zeocin®

Growth medium: DMEM, 2 mM L-glutamine, 4.5 g/l glucose, 10% FBS supplemented with 100 µg/ml Normocin™

Quality Control:

• Biallelic TLR4 knockout has been verified by DNA sequencing and functional assays.
• The biallelic replacement of the mouse MIP-2 (macrophage inflammatory protein-2; CXCL2; murine homolog of IL-8) open reading frame (ORF) with the SEAP reporter ORF has been verified by PCR and sequencing. Furthermore, the inability to produce MIP-2 has been confirmed by ELISA.
• Activity of RAW-Dual™ KO-TLR4 cells has been tested with pattern recognition receptor (PRR) ligands that trigger the NF-κB and interferon regulatory factor (IRF) signaling pathways.
• Cell line stability for 20 passages following thawing has been verified.
• RAW-Dual™ KO-TLR4 cells are guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ of RAW-Dual™ KO-TLR4 cells in a cryovial or shipping flask
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi.
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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

Details

Toll‑like receptor 4 (TLR4) is the receptor for Gram-negative lipopolysaccharide (LPS) and its toxic moiety called lipid A. TLR4 interacts with three different extracellular proteins: the LPS-binding protein (LBP), CD14, and the myeloid differentiation protein 2 (MD‑2). Their interaction induces a signaling cascade resulting in the activation of NF-κB and the production of proinflammatory cytokines. LPS contamination is a common issue arising when testing novel compounds, such as recombinant proteins, leading to unreliable results. TLR4 knockout cells ensure the study of PRR pathways without interference from LPS.