Mouse TLR3 Reporter HEK 293 Cells

NF-κB-SEAP reporter cells

ABOUT

NF-κB–SEAP reporter HEK293 cells expressing murine TLR3

HEK-Blue™ mTLR3 cells were engineered from the human embryonic kidney HEK293 cell line to study the murine Toll-like receptor 3 (mTLR3)-dependent NF-κB response. This important pattern recognition receptor (PRR) recognizes double-stranded (ds)RNA, a hallmark of viral replication, and triggers antiviral NF-κB and IRF immune responses [1].

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Description

HEK-Blue™ mTLR3 cells feature the stable expression of the murine TLR3 gene as well as an inducible reporter gene for SEAP (secreted embryonic alkaline phosphatase). SEAP (secreted embryonic alkaline phosphatase) levels produced upon TLR3 stimulation can be readily determined by performing the assay in HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP. Alternatively, SEAP activity may be monitored using QUANTI-Blue™, a SEAP detection reagent.

HEK-Blue™ mTLR3 cells are highly responsive to synthetic analogs of dsRNA. They show potent NF-κB response upon incubation with TLR3-specific ligands, such as Poly(I:C) (polyinosinic-polycytidylic acid) or Poly(A:U) (polyadenylic–polyuridylic acid) (see figures)

Of note, HEK293 cells express endogenous levels of various human PRRs, including TLR3, TLR5, and RIG-I-like receptors, and therefore might respond to their cognate ligands (see figures)

 

Key features

  • Stable expression of mouse TLR3
  • Strong response to dsRNA and analogs
  • Distinct monitoring of TLR3-dependent NF-κB activation by assessing the SEAP activities

Applications

  • Defining the role of murine TLR3-dependent NF-κB signaling pathway
  • Screening for novel TLR3 agonists and inhibitors in comparison with their parental cell line HEK-Blue™ Null1-k


References

1. Chen Y, et al., 2021.  Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J Zhejiang Univ Sci B.;22(8):609-632. 

Disclaimer:  These cells are for internal research use only and are covered by a Limited Use License (See Terms and Conditions). Additional rights may be available.

SPECIFICATIONS

Specifications

Species
Mouse
Tested applications

Defining the role of murine TLR3-dependent NF-κB signaling pathway, Screening for novel TLR3 agonists and inhibitors

Cell type
Epithelial
Growth properties
Adherent
Tissue origin
Human embryonic kidney cells
Reporter gene
SEAP
Detection method
Colorimetric
Antibiotic resistance
Blasticidin
Zeocin®
Growth medium

Complete DMEM (See TDS)

Mycoplasma-free

Verified using Plasmotest™

Quality control

Each lot is functionally tested and validated. 

CONTENTS

Contents

  • Product: 
    HEK-Blue™ mTLR3 Cells
  • Cat code: 
    hkb-mtlr3
  • Quantity: 
    3-7 x 10^6 cells
Includes:

1 ml of Blasticidin (10 mg/ml) 1 ml of Zeocin® (100 mg/ml) 1 ml of Normocin® (50 mg/ml) 1 pouch of HEK-Blue™ Detection

Shipping & Storage

  • Shipping method:  Dry ice
  • Storage:

    • Liquid Nitrogen Vapor
    Stability: 20 passages

    Caution:

    • Upon receipt, store immediately in liquid nitrogen vapor. Do not store cell vials at -80°C.

Details

Toll-Like Receptor 3

The Toll-Like Receptor (TLR) family members TLR3, TLR7, TLR8, and TLR9 are specialized in sensing viral-derived components and are mainly found in the endosome. Among these, TLR3 recognizes double-stranded (ds)RNA, a hallmark of viral replication, and triggers antiviral immune responses [1]. TLR3 is expressed in myeloid dendritic cells, macrophages, as well as non-immune cells [2].

TLR3 signaling

TLR3 activation upon viral infection involves several steps, including translocation of TLR3 from the ER (endoplasmic reticulum) via the Golgi to the endosome, proteolytic cleavage and dimerization of TLR3, and finally receptor-ligand binding [3]. In order to start the signaling cascade, activated TLR3 recruits the adaptor protein TRIF (TIR domain-containing adapter-inducing interferon-β). TRIF binds to TRAF3 (TNF receptor-associated factor 3), which then recruits TBK1 (TANK-binding kinase 1) and IKKε (IκB kinase ε), thus activating the transcription factor IRF3 (interferon regulatory factor 3) and stimulating the production of type I IFNs (interferons). Additionally, TRIF interacts with TRAF6 and RIP1 (kinase receptor-interacting protein 1). RIP1 in turn binds to TAK1 (transforming growth factor β-activated kinase 1) and IKK. TAK1 phosphorylates IKKα and IKKβ, leading to the phosphorylation of IκB, the NF-κB inhibitor. Ultimately, this leads to the release and translocation of NF-κB into the nucleus and the induction of pro-inflammatory cytokines [2,4]. 

Pathology

Given its important role in dsRNA recognition, TLR3 signaling has been intensively studied. Various TLR3-agonists, such as the synthetic dsRNA analog Poly(I:C) are being used in vaccine development and cancer therapy [4]. Yet, recent studies have indicated that TLR3 may act as a double-edged sword by showing both protective and damaging functions in the context of some human viral infections [3,5]. Moreover, rare mutations in TLR3 have been associated with viral susceptibility; specifically, infections with HSV-1 (herpes simplex virus 1), influenza, and SARS-Co-V2 have been linked to pathogenic germline variants in TLR3 pathway genes [2]. Understanding the TRIF-dependent TLR3 pathway may be essential for the establishment of specific therapeutic approaches to diminish TLR3-driven disease and exploit its protective functions [3].

 

 

References

1. Manuela Sironi, et al., 2012. A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection. J Immunol 15; 188 (2): 818–823. 
2. Aluri, J, et al., 2021. Toll-Like Receptor Signaling in the Establishment and Function of the Immune System. Cells, 10, 1374.
3. Chen Y, et al., 2021.  Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J Zhejiang Univ Sci B.;22(8):609-632.
4. Komal A, et al., 2021. TLR3 agonists: RGC100, ARNAX, and poly-IC: a comparative review. Immunol Res. 69(4):312-322. 
5. Perales-Linares R, Navas-Martin S. 2013. Toll-like receptor 3 in viral pathogenesis: friend or foe? Immunology.;140(2):153-67.

DOCUMENTS

Documents

HEK-Blue™ mTLR3 Cells

Technical Data Sheet

Validation Data Sheet

Safety Data Sheet

Certificate of analysis

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