THP1-Dual™ KO-MDA5 Cells

KO-MDA5 dual reporter monocytes for RLR pathway studies

Reporter systems in THP1-Dual™-derived cells

InvivoGen offers a new series of THP1-Dual™ cell lines,  derived from the human THP-1 monocytic cell line, and specifically designed for assessing the role of RIG-I, MDA5, and MAVS in the cytosolic RNA sensing pathways. 

THP1-Dual™ KO-MDA5 cells express two inducible reporter genes, allowing the concomitant study of the IRF and NF-κB pathways, by monitoring the Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase) activities, respectively. In addition, these cells feature a stable knockout of the MDA5 ((Melanoma Differentiation Associated gene 5) gene.

RIG-I and MDA5 are two distinct sensors of viral double-stranded RNA (dsRNA), a replication intermediate for RNA viruses [1-3]. Upon recognition of dsRNA, RIG-I and MDA5 are recruited by the MAVS adaptor to the outer membrane of the mitochondria leading to the activation of several transcription factors, including interferon regulatory factors (IRFs) and NF-κB [3].

More details

KEY FEATURES

• Verified knockout of the MDA5 gene (PCR, DNA sequencing, Western blot, and functional assays)
• Functionally validated with a selection of PRR ligands and cytokines
• Readily assessable Lucia luciferase and SEAP reporter activities
• The stability for 20 passages, following thawing, has been verified
• Guaranteed mycoplasma-free

APPLICATIONS

• Defining the role of MDA5 in cytosolic RNA sensing pathways
• Highlighting possible overlap between RIG-I/MDA5 and STING signaling functions
• Developing novel specific inhibitors of the RLR signaling pathway

References

1. Gebhardt A. et al., 2017. Discrimination of Self and Non-Self Ribonucleic Acids. Journal of Interferon & Cytokine Research 37: 184-97.
2. Pichlmair A. et al., 2006. RIG-I mediated antiviral responses to single-stranded RNA bearing 5’-phosphates. Science 314:997-1001.
3. 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.

Figures

Validation of MDA5 KO.
(A) The targeted MDA5 region in THP1-Dual™ (WT; blue arrow) parental cells and THP1-Dual™ KO-MDA5 (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-MDA5 cells feature a frameshift deletion, causing an early stop codon and inactivation of MDA5.  (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-MMDA5(KO) cells were analyzed using an anti-human MDA5 antibody (green arrow), followed by a HRP-conjugated anti-rabbit secondary antibody (WES assay). As expected, a band was detected at ~135 KDa in the WT cells only.

IRF responses in THP1-Dual™-derived cells.
THP1-Dual™ (WT) and THP1-Dual™ KO-MDA5 cells were incubated with 1 ng/ml human TNF-α (hTNF-α), or 10⁴ U/ml human IFN-β (hIFN-β), 300 ng/ml 3p-hpRNA/Lyovec, 1 μg/ml Poly(I:C) HMW/LTX, 1 μg/ml VACV-70/Lyovec, 3 μg/ml 2’3’-cGAMP, 3 ng/ml Pam3CSK4, or 1 μg/ml R848. After overnight incubation, the IRF response was assessed by measuring Lucia luciferase activity in the supernatant using QUANTI-Luc™. Data are shown as a fold change (mean ± SEM) over non-induced cells.

 NF-κB responses in THP1-Dual™- derived cells.
THP1-Dual™ (WT) and THP1-Dual™ KO-MDA5 cells were incubated with 1 ng/ml human TNF-α (hTNF-α), or 10⁴ U/ml human IFN-β (hIFN-β), 300 ng/ml 3p-hpRNA/Lyovec, 1 μg/ml Poly(I:C) HMW/LTX, 1 μg/ml VACV-70/Lyovec, 3 μg/ml 2’3’-cGAMP, 3 ng/ml Pam3CSK4, or 1 μg/ml R848. After overnight incubation, the NF-κB activity was assessed by measuring the SEAP activity in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

Specifications

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Antibiotic resistance: Blasticidin and Zeocin®

Quality Control:

• Biallelic MDA5 knockout has been verified by PCR, DNA sequencing, and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Contents

• 3-7 x 10⁶ cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 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

MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard)

MDA5 is a cytoplasmic RNA helicase that plays an important role in antiviral response. It senses long double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, leading to the production of type I interferons (IFNs) [1]. MDA-5 and the related RNA helicase RIG-I recognize a complementary set of cytosolic viral dsRNA. Transfected Poly(I:C), a synthetic analog of viral dsRNA, is recognized by both MDA-5 and RIG-I.

RIG-I (retinoic-acid-inducible protein 1, also known as Ddx58)

RIG-I is a cytoplasmic RNA helicase that is critical for host antiviral responses. It senses double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, leading to the production of type I interferons (IFNs) [1]. RIG-I binds specifically to short dsRNAs that have blunt ends and a 5’-triphosphate (5’-ppp) moiety, facilitating discrimination between host and viral dsRNA [2].

MAVS (mitochondrial antiviral-signaling protein, also known as IPS‑1, CARDIF, VISA)

MAVS is an adaptor protein that plays a critical role in the immune response to viral infection. The innate immune system senses intracellular double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, through two RNA helicases: retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5). Upon recognition of dsRNA, RIG-I and MDA5 are recruited by MAVS 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. IRFs and NF-κB regulates the expression of type I interferons (IFNs) and pro-inflammatory cytokines, respectively [1, 3].

1. Gebhardt A. et al., 2017. Discrimination of Self and Non-Self Ribonucleic Acids. Journal of Interferon & Cytokine Research 37: 184-97.
2. Pichlmair A. et al., 2006. RIG-I mediated antiviral responses to single-stranded RNA bearing 5’-phosphates. Science 314:997-1001.
3. 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.

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