Human MDA5 Dual Reporter HEK 293 Cells (TLR3 & RIG-I deficient)

NF-κB-SEAP and IRF-Lucia reporter cells

ABOUT

HEK-Dual™ RNA-Null cells expressing human MDA5

HEK-Dual™ RNA-hMDA5 cells are designed to exclusively assess the role of the human melanoma differentiation-associated protein 5 (MDA5) 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 Toll-like receptor 3 (TLR3), Retinoic acid-inducible protein 1 (RIG-I), and MDA5.

In HEK-Dual™ RNA-hMDA5 cells, human MDA5 has been reintroduced, allowing the study of MDA5-specific responses without interference from RIG-I and TLR3 (see figures).

More details More details

 

Key features

  • Strong MDA5 responses without TLR3 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.

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
Human
Target

MDA5

Target species

Human

Tested applications

MDA5 activation cellular assays

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

Complete DMEM (see TDS)

Mycoplasma-free

Validated using Plasmotest™

Quality control

Each lot is functionally tested and validated.

CONTENTS

Contents

  • Product: 
    HEK-Dual™ RNA-hMDA5 Cells
  • Cat code: 
    hkd-rna-mda5
  • Quantity: 
    3-7 x 10^6 cells
Includes:
  • 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).
  • 1 ml of QB reagent and 1 ml of QB buffer
  • 1 tube of QUANTI-Luc™ 4 Reagent

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

Cell line description

HEK-Dual™ RNA-hMDA5 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-hMDA5 cells, human MDA5 has been reintroduced.

Therefore, strong NF-κB and/or IRF responses can be observed upon stimulation with complexed Poly(I:C) (HMW). As expected, HEK-Dual™ RNA-hMDA5 cells do not respond to the cognate ligands of RIG-I or TLR3 (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. 

CUSTOMER SERVICE & TECHNICAL SUPPORT

Question about this product ?