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THP1-Dual™ KO-TBK1 Cells

THP1-Dual™ KO-TBK1 Cells Unit size Cat. code Docs Qty Price
Human THP-1 Monocytes - TBK1 knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
thpd-kotbk
+-
$1,421.00

NEW PRODUCT

TBK1 knockout dual reporter monocytes

Nucleic acid sensing signaling pathways in THP1-Dual™ cells
Nucleic acid sensing signaling pathways in THP1-Dual™ cells

THP1-Dual™ KO-TBK1 cells were generated from the THP1-Dual™ cell line, which is derived from the human THP-1 monocytes, through the stable knockout of the TBK1 gene. TBK1 (TANK-binding kinase 1) functions as a key node protein in several cell signaling pathways, including innate immune responses elicited upon pattern recognition receptor (PRR) activation. Upon ligand (e.g. cytoplasmic nucleic acids) binding, these receptors trigger the production of interferons (IFNs) through the activation of the TBK1/IKKε-IRF3/IRF7 (interferon regulatory factors 3 and 7) pathway [1]. Activated PRRs also trigger the production of pro-inflammatory cytokines through activation of the NF-κB pathway [1]. 

THP1-Dual™ KO-TBK1 and THP1-Dual™ cells feature two reporter genes allowing the simultaneous study of the IRF pathway, by monitoring the activity of an inducible secreted Lucia luciferase, and the NF-κB pathway by monitoring the activity of an inducible SEAP (secreted embryonic alkaline phosphatase). Lucia luciferase and SEAP activities are readily assessable in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution detection reagents, respectively.

As expected, the responses of THP1-Dual™ KO-TBK1 cells to the cyclic dinucleotide 2'3'-cGAMP (a STING agonist) and to lipopolysaccharide (a TLR4 agonist) are impaired. However, differential responses (either no effect, decrease or increase) are observed when using RNA or DNA agonists coupled with transfection reagents. THP1-Dual™ KO-TBK1 cells retain the ability to respond to cytokines such as type I IFNs and TNF-α. These cells are resistant to Blasticidin and Zeocin™.

 

Features of THP1-Dual™ KO-TBK1 cells:

  • Biallelic knockout of the TBK1 gene
  • Readily assessable Lucia luciferase and SEAP reporter activity

Applications for THP1-Dual™ KO-TBK1 cells:

  • Defining the role of TBK1 in PRR-induced signaling, or other cell signaling pathways (e.g. cell growth)
  • Highlighting possible overlapping PRR activation or regulatory mechanisms (see 'Details' tab)

Validation of THP1-Dual™ KO-TBK1 cells:

  • TBK1 knockout verified by PCR, DNA sequencing, and western-blot
  • Functionally tested

 

References:

1. Iurescia S. et al., 2018. Nucleic acid sensing machinery: targeting innate immune system for cancer therapy. Recent Pat. Anticancer Drug Discov. 13: 2-17

Figures

Validation of TBK1 knockout by western blot (WES by Protein Simple)
Validation of TBK1 knockout by western blot (WES by Protein Simple)

Analysis of lysates from the THP1-Dual™ (WT) and THP1-Dual™ KO-TBK1 (KO) cells using Anti-TBK1 (10 μg/ml), followed by HRP-conjugated anti-mouse secondary antibody (undiluted). The arrow indicates the expected band for the TBK1 protein (83 KDa).

2’3’-cGAMP-induced IRF and NF-κB responses in THP1-Dual-derived cells
2’3’-cGAMP-induced IRF and NF-κB responses in THP1-Dual-derived cells

2x105 THP1-Dual™ (WT) and THP1-Dual™ KO-TBK1 (KO-TBK1) cells were incubated with 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, IRF (A) and NF-κB (B) responses were assessed by measuring the activity of Lucia luciferase and SEAP in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution, respectively. Activity fold increase over non-induced cells (Lucia luciferase readout) or reading of optical density (OD) at 630 nm (SEAP readout) are shown.

RNA-induced IRF and NF-κB responses in THP1-Dual-derived cells
RNA-induced IRF and NF-κB responses in THP1-Dual-derived cells

2x105 THP1-Dual™ or THP1-Dua™ KO-TBK1 cells were transfected with 1 μg/ml 3p-hpRNA or 1 μg/ml 5’ppp-dsRNA complexed with LyoVec™ (A, B) or LTX (C, D). After overnight incubation, IRF response was assessed by measuring bioluminescent activity of the Lucia luciferase in the supernatant using QUANTI-Luc™. Activity fold increase over non-transfected cells is shown (A, C). The NF-κB activity in THP1-Dual™-derived cells was assessed by measuring the SEAP activity in the supernatant using QUANTI-Blue™ Solution. Reading of optical density (OD) at 630 nm is shown (B, D).

Cytokine or LPS-induced IRF and NF-κB responses in THP1-Dual-derived cells
Cytokine or LPS-induced IRF and NF-κB responses in THP1-Dual-derived cells

2x105 THP1-Dua™ or THP1-Dual™ KO-TBK1 cells were incubated with 10 ng/ml human TNF-α (hTFN-α), 104 U/ml human IFN-α (hIFN-α), 104 U/ml human IFN-β (hIFN-β), or 1 μg/ml LPS EK Ultrapure (UP). After overnight incubation, IRF (A) and NF-κB (B) responses were assessed by measuring the activity of Lucia luciferase and SEAP in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution, respectively. Activity fold increase over non-induced cells (Lucia luciferase readout) or reading of optical density (OD) at 630 nm (SEAP readout) are shown.

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Specifications

Antibiotic resistance: Blasticidin and Zeocin™

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™

Quality Control:

  • Biallelic TBK1 knockout has been verified by PCR, DNA sequencing, western blot, and functional assays.
  • The stability for 20 passages, following thawing, has been verified. 
  • These cells are guaranteed mycoplasma-free. 

This product is covered by a Limited Use License (See Terms and Conditions).

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Contents

  • 1 vial of THP1-Dual™ KO-TBK1 cells (3-7 x 106 cells) in freezing medium
  • 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
  • 1 ml of Zeocin™ (100 mg/ml)
  • 1 ml of Blasticidin (10 mg/ml)
  • 1 pouch of QUANTI-Luc™
  • 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

 Shipped on dry ice (Europe, USA & Canada)

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Details

THP1-Dual™ KO-TBK1 cells have been functionally tested using various sources of nucleic acids (see the validation data document).

As expected, IRF and NF-κB responses are severely impaired when the THP1-Dual™ KO-TBK1 cells are incubated with STING agonists, such as 2’3’-cGAMP, which do not require transfection to access the cytosol. However, differential responses are observed when using RNA agonists with transfection reagents. The weak RIG-I agonist 5’ppp-dsRNA loses its ability to induce an IRF response in KO-TBK1 cells when complexed with LyoVec™ or LTX. On the contrary, the IRF response is unexpectedly increased when using the highly potent RIG-I agonist 3p-hpRNA complexed to LyoVec™ or LTX. Surprisingly, with either agonist, the NF-κB response is barely affected, if not slightly increased. These data suggest that the use of different agonists and transfection reagents could highlight overlapping RNA-sensing or regulatory mechanisms [1, 2].

 

References:

1. Perry AK. et al., 2004. Differential requirement for TANK-binding kinase-1 in type I interferon responses to Toll-like receptor activation and viral infection. J. Exp. Med. 199:1651.
2. Deng W. et al., 2008. Negative regulation of virus-triggered IFN-B signaling pathway by alternative splicing of TBK1. J. Biol. Chem. 283:35590. 

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