Specific TLR3 Reporter HEK293 Cells (RIG-I & MDA5 deficient)

Notification: Reference #hkd-rna-tlr3-av can only be ordered together with reference #hkd-rna-tlr3.

HEK-Dual™ RNA-Null cells expressing human TLR3

Signaling pathways in HEK-Dual™ RNA-hTLR3 cells

HEK-Dual™ RNA-hTLR3 cells are designed to exclusively assess the role of the human Toll-like receptor 3 (TLR3) in double-stranded (ds)RNA signaling. This colorimetric and luminescent bioassay can be used to find novel anti‐viral therapies or effective mRNA vaccines.
 

This cell line derives from the HEK-Dual™ RNA-Null cells, which express two reporter proteins, an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter and an interferon regulatory factor (IRF)-inducible Lucia® luciferase reporter. Furthermore, they have no endogenous expression of three important dsRNA sensors Melanoma differentiation-associated gene 5 (MDA5), Retinoic acid-inducible protein 1 (RIG-I), and TLR3.

In HEK-Dual™ RNA-hTLR3 cells, human TLR3 has been reintroduced, allowing the study of TLR3-specific responses without interference from RIG-I and MDA5 (see figures). 

 More details

Key features

• Strong TLR3 responses without MDA5 and RIG-I interference
• Simultaneously assessable NF-κB-SEAP and IRF-Lucia® reporter activity
• Convenient readout using QUANTI-Blue™ and QUANTI-Luc™ 4 Lucia/Gaussia
• Stability guaranteed for 20 passages

Applications

• Comparable RNA sensor studies
• Drug screening
• mRNA-based and anti-viral vaccine development

Upon recognition of viral or synthetic dsRNA, the major pattern recognition receptors (PRRs) MDA5, RIG-I, and TLR3 collectively establish an antiviral host response, by mediating the transcriptional induction of type I interferons (IFNs) and proinflammatory cytokines or even promoting cell death. The deep insights of these RNA PRRs can be utilized to find small‐molecular agonists for anti‐viral therapy and effective vaccine strategies.

InvivoGen’s products are for research use only, and not for clinical or veterinary use.

Figures

Human TLR3 expression in HEK-Dual™-derived cells. Lysates from HEK-Dual™ RNA-Null (Null) and HEK-Dual™ RNA-hTLR3 cells (TLR3) were analyzed using an anti-human TLR3 antibody, followed by an HRP-conjugated secondary antibody (WESS™).

NF-κB and IRF dose responses of HEK-Dual™ RNA-hTLR3 cells to TLR3 ligands. Cells were stimulated for 24h with increasing concentrations of Poly(I:C) HMW ± complexed with LyoVec™ (LV) or NexaVant®. The NF-κB-induced SEAP activity was assessed using QUANTI-Blue™ (1h incubation). Data are shown as optical density (OD) at 650 nm (mean ± SEM). The IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™ 4 Lucia/Gaussia. Data are shown in fold response over non-induced cells (mean ± SEM).

NF-κB responses of HEK-Dual™ RNA-Null and HEK-Dual™ RNA-hTLR3 cells to PRR agonists and cytokines. Cells were stimulated for 24h with various cytokines and PRR agonists: Human (h)TNF-α (NF-κB-positive control, 10 ng/ml), Poly(I:C) high or low molecular weight (HMW/LMW), NexaVant® (TLR3 ligands, 1 µg/ml), Poly(I:C) HMW/LMW complexed with LyoVec™ (LV) (RLR ligands, 1 µg/ml), and 3p-hpRNA/LV (RIG-I ligand, 1 µg/ml). The NF-κB-induced SEAP activity was assessed using QUANTI-Blue™ (1h incubation). Data are shown as optical density (OD) at 650 nm (mean ± SEM).

IRF responses of HEK-Dual™ RNA-Null and HEK-Dual™ RNA-hTLR3 cells to PRR agonists and cytokines. Cells were treated as described before. Human (h)IFN-β (30 U/ml) was used as an IRF-positive control. After 24h incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™ 4 Lucia/Gaussia. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: TLR3

Specificity: Human

Reporter gene: SEAP, Lucia®

Antibiotic resistance: Blasticidin, Puromycin, Zeocin®

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

All of these products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶  cells in a cryovial or shipping flask.
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Puromycin (100 mg/ml)
• 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

Cell line description

HEK-Dual™ RNA-hTLR3 cells derived from the HEK-Dual™ RNA-Null cell line. These cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter and an interferon regulatory factor (IRF)-inducible Lucia® luciferase reporter. This allows the simultaneous study of the NF-κB pathway, by monitoring the activity of SEAP, and the IRF pathway, by assessing Lucia® luciferase activity. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent. 

The parental cell line HEK-Dual™ RNA-Null lacks three critical double-stranded (ds)RNA sensors — Melanoma Differentiation Associated gene 5 (MDA5), Retinoic Acid Inducible protein 1 (RIG-I), and Toll-like receptor 3 (TLR3). In HEK-Dual™ RNA-hTLR3 cells, human TLR3 has been reintroduced.

Therefore, strong NF-κB and/or IRF responses can be observed upon stimulation with naked or complexed Poly(I:C) (HMW). As expected, HEK-Dual™ RNA-hTLR3 cells do not respond to the cognate ligands of RIG-I or MDA5 (see figures). 

RNA sensor background

To combat viral infection and evasion mechanisms, nature has implemented a multitude of partially overlapping defense strategies. The antiviral response is initiated through the recognition of viral products, such as double-stranded (ds) RNA, by two types of pathogen recognition receptors (PRRs) [1]:

• the RIG-I-like receptors (RLRs) and
• the Toll-like receptors (TLRs).

MDA5 & RIG-I

MDA5 (Melanoma-differentiation-associated gene 5, MDA-5, IFIH1 or Helicard) and RIG-I (retinoic-acid-inducible protein 1, also known as Ddx58) are cytoplasmic RNA helicases belonging to the RLR family. Both sense dsRNA, a replication intermediate of RNA viruses, leading to the production of type I interferons (IFNs) [1]. They recognize a complementary set of cytosolic viral dsRNA. MDA5 recognizes long dsRNA, and accordingly senses the single positive RNA viruses such as the poliovirus. RIG‐I prefers short dsRNA ligands and specifically recognizes most single‐negative RNA viruses which generate lots of short 5′ ppp‐dsRNA during replication (e.g Influenza). Additionally, it is able to sense positive single RNA viruses such as the hepatitis C virus. It was also shown that RIG-I can detect certain DNA viruses and bacteria. On the other hand, both RIG‐I and MDA5 cross‐detect the same viruses, including rota and corona viruses. The synthetic analog of viral dsRNA, transfected Poly(I:C), is also recognized by both sensors [4]. Upon viral infection, RIG-I and MDA5 are recruited by the adaptor protein MAVS (Mitochondrial antiviral-signaling protein) to the outer membrane of the mitochondria leading to the activation of several transcription factors including interferon-regulatory factor 3 (IRF3), IRF7, and NF-κB. Subsequently, IRFs and NF-κB regulate the expression of type I interferons (IFNs) and pro-inflammatory cytokines, respectively [1-3].

TLR3

Within the large family of TLRs, TLR3 is specialized in sensing viral-derived components and is mainly found in the endosome [4]. Its activation upon viral infection involves several steps, including translocation from the ER (endoplasmic reticulum) to the endosome, proteolytic cleavage and dimerization of TLR3, and finally receptor-ligand binding [6]. 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) and TRAF6, activating the transcription factor IRF3 and NF-κB, respectively.  Ultimately, this leads to the production of type I IFNs (interferons) and pro-inflammatory cytokines [5,7].

References

1. Kawai T. et al., 2005. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol. 6(10):981-988.
2. Gebhardt A. et al., 2017. Discrimination of Self and Non-Self Ribonucleic Acids. Journal of Interferon & Cytokine Research 37: 184-97.
3. Pichlmair A. et al., 2006. RIG-I mediated antiviral responses to single-stranded RNA bearing 5’-phosphates. Science 314:997-1001.
X. Vabret N, Blander JM. Sensing microbial RNA in the cytosol. Front Immunol. 2013 Dec 25;4:468.
4. Manuela Sironi, et al., 2012. A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection. J Immunol 15; 188 (2): 818–823. 
5. Aluri, J, et al., 2021. Toll-Like Receptor Signaling in the Establishment and Function of the Immune System. Cells, 10, 1374.
6. 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.
7. Komal A, et al., 2021. TLR3 agonists: RGC100, ARNAX, and poly-IC: a comparative review. Immunol Res. 69(4):312-322. 

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