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THP1-Dual™ KO-RIG-I / KO-MDA5 / KO-MAVS

THP1-Dual™ KO-RIG-I Cells Unit size Cat. code Docs Qty Price
Human THP-1 Monocytes - RIG-I knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
thpd-korigi
+-
$1,515.00
THP1-Dual™ KO-MDA5 Cells Unit size Cat. code Docs Qty Price
Human THP-1 Monocytes - MDA5 knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
thpd-komda5
+-
$1,515.00
THP1-Dual™ KO-MAVS Cells Unit size Cat. code Docs Qty Price
Human THP-1 Monocytes - MAVS knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
thpd-komavs
+-
$1,515.00

Dual reporter monocytes for RLR pathway studies

Reporter systems in THP1-Dual™-derived cells
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-RIG-I cells
  - THP1-Dual™ KO-MDA5 cells

  - THP1-Dual™ KO-MAVS cells

These 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 stable knockout of either the RIG-I (Retinoic Acid Inducible protein 1), MDA5 (Melanoma Differentiation Associated gene 5), or MAVS (Mitochondrial antiviral-signaling protein) genes.

 

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 RIG-I, MDA5, or MAVS genes (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 RIG-I, MDA5, or MAVS 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 RIG-I knockout
Validation of RIG-I knockout

Validation of RIG-I KO.
(A)
The targeted MAVS region in THP1-Dual™ (WT; blue arrow) parental cells and THP1-Dual™ KO-RIG-I (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-RIG-I cells feature a frameshift deletion, causing an early stop codon and inactivation of RIG-I.
(B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-RIG-I (KO) cells were analyzed using an anti-human RIG-I antibody (green arrow), followed by a HRP-conjugated anti-rabbit secondary antibody (WES assay). As expected, a band was detected at ~102 Da in the WT cells only.

Functional validation of RIG-I knockout (IRF response)
Functional validation of RIG-I knockout (IRF response)

IRF responses in THP1-Dual™-derived cells.
THP1-Dual™ (WT) and THP1-Dual™ KO-RIG-I 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.

Functional validation of RIG-I knockout (NF-κB response)
Functional validation of RIG-I knockout (NF-κB response)

NF-κB responses in THP1-Dual™-derived cells.
THP1-Dual™ (WT) and THP1-Dual™ KO-RIG-I 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).

Validation of MDA5 knockout
Validation of MDA5 knockout

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.

Functional validation of MDA5 knockout (IRF response)
Functional validation of MDA5 knockout (IRF response)

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.

Functional validation of MDA5 knockout (NF-κB response)
Functional validation of MDA5 knockout (NF-κB response)

 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).

Validation of MAVS knockout
Validation of MAVS knockout

Validation of MAVS KO.
(A) The targeted MAVS region in THP1-Dual™ (WT; blue arrow) parental cells and THP1-Dual™ KO-MAVS (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-MAVS cells feature a frameshift deletion, causing an early stop codon and inactivation of MAVS. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-MAVS (KO) cells were analyzed using an anti-human MAVS antibody (green arrow), followed by a HRP-conjugated anti-rabbit secondary antibody (WES assay). As expected, a band was detected at ~80 Da in the WT cells only.


IRF responses in THP1-Dual-derived cells.
THP1-Dual
(WT) and THP1-Dual KO-MAVS 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.

Functional validation of MAVS knockout (NF-κB response)
Functional validation of MAVS knockout (NF-κB response)

NF-κB responses in THP1-Dual™-derived cells.
THP1-Dual™ (WT) and THP1-Dual™ KO-MAVS 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).

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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 RIG-I, MDA5, or MAVS 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. 

 

InvivoGen's products are covered by a Limited Use License (See Terms and Conditions).

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Contents

Please note: Each cell line is sold separately. See TDS for the exact contents of each cell line. 

  • 3-7 x 106 THP1-Dual™ KO-RIG-I cells, OR THP1-Dual™ KO-MDA5 cells, OR THP1-Dual™ KO-MAVS 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 pouch of QUANTI-Luc™

 Shipped on dry ice (Europe, USA & Canada)

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Details

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].

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.

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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FAQ

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

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