Control RNA Sensor Reporter HEK293 Cells (NF-κB and IRF)

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

TLR3/RIG-I/MDA5 deficient IRF and NF-κB reporter cell line

Engineered HEK-Dual™ RNA-Null cells

HEK-Dual™ RNA-Null cells are a control cell line designed to assess the double-stranded (ds)RNA sensors TLR3, RIG-I, and MDA5. This colorimetric and luminescent bioassay can be used to find novel anti‐viral therapies or effective mRNA vaccines.
 

This cell line expresses two reporter proteins, an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter and an interferon regulatory factor (IRF)-inducible Lucia® luciferase reporter. Furthermore, HEK-Dual™ RNA-Null cells have no endogenous expression of three important dsRNA sensors Toll-like receptor 3 (TLR3), Retinoic acid-inducible protein 1 (RIG-I), and Melanoma differentiation-associated gene 5 (MDA5).

 More details

As a result, they do not respond to any synthetic dsRNA analog compared to their parental cell line HEK-Dual™ (see figures). 

Key features

• Deficient in TLR3, RIG-I, and MDA5 responses
• 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

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

HEK-Dual™ RNA-Null cells allow for the independent study of one important dsRNA-sensor when used in combination with:

–  HEK-Dual™ RNA-hTLR3 cells
–  HEK-Dual™ RNA-hRIG-I cells
–  HEK-Dual™ RNA-hMDA5 cells

Upon recognition of viral or synthetic dsRNA, the major pattern recognition receptors (PRRs) TLR3, RIG-I, and MDA5 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, RIG-I, and MDA5 expression in HEK-Dual™-derived cells. Lysates from HEK-Dual™ (WT) and HEK-Dual™ RNA-Null cells (Null) were analyzed using an anti-human TLR3, RIG-I, or MDA5 antibody, followed by an HRP-conjugated secondary antibody (WESS™).

NF-κB response of HEK-Dual™ RNA-Null cells to PRR agonists and cytokines. HEK-Dual™ RNA-Null 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 molecular weight (HMW) (TLR3 ligand, 10 µg/ml), Poly(I:C) HMWcomplexed with LyoVec™ (LV) (RIG-I/MDA5 ligand, 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 response of HEK-Dual™ RNA-Null cells to PRR agonists and cytokines. HEK-Dual™ RNA-Null 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: MDA5, RIG-I, TLR3

Specificity: Human

Reporter gene: SEAP, Lucia®

Antibiotic resistance: Blasticidin, 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

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 ml Zeocin® (100 mg/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)
• 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-Null 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. These modifications allow 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. 

This cell line 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) — making them a great tool for studying RNA sensor signaling, vaccine development, and anti-viral response mechanisms. 

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) [1,2]:

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

Upon recognizing viral or synthetic dsRNA, these major PRRs mediate the transcriptional induction of proinflammatory cytokines, chemokines, and type I interferons (IFNs), thereby inducing hundreds of interferon-stimulated genes (ISGs). These products have antiviral, immunomodulatory, cell growth regulatory, and metabolic regulatory actions that create an antiviral state. If successful, this response potently restricts virus replication and cell-to-cell spread of infection [3]. 

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 [4]. MDA5 recognizes long dsRNA and accordingly senses the single positive RNA viruses such as the poliovirus. On the contrary, 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 can 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. Both RIG‐I and MDA5 cross‐detect the same viruses, including rota and coronaviruses. 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 [1,5].

TLR3

Within the large family of TLRs, TLR3 is specialized in sensing viral-derived components and is mainly found in the endosome [6]. It recognizes primarily genomic dsRNA of reoviruses, as well as the intermediate RNAs generated during the replication of different viruses including herpes simplex virus‐1, and enteroviruses [3]. 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 [7]. 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 [8-9].

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. Qiao, H, et al., 2021. Cell fate determined by the activation balance between PKR and SPHK1. Cell Death Differ 28, 401–418.
3. Kell AM, Gale M Jr., 2015. RIG-I in RNA virus recognition. Virology.;479-480:110-21.
4. Chen N, et al., 2017. RNA sensors of the innate immune system and their detection of pathogens. IUBMB Life.;69(5):297-304. 
5. Pichlmair A. et al., 2006. RIG-I mediated antiviral responses to single-stranded RNA bearing 5’-phosphates. Science 314:997-1001.
6. Manuela Sironi, et al., 2012. A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection. J Immunol 15; 188 (2): 818–823. 
7. 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.
8. Aluri, J, et al., 2021. Toll-Like Receptor Signaling in the Establishment and Function of the Immune System. Cells, 10, 1374.
9. Komal A, et al., 2021. TLR3 agonists: RGC100, ARNAX, and poly-IC: a comparative review. Immunol Res. 69(4):312-322. 

FAQ Cell Lines

Any questions about our cell lines ? Visit our frequently asked questions page