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THP1-Dual™ KI-hSTING-M155 Cells [ NEW ]

THP1-Dual™ KI-hSTING-M155 Cells Unit size Cat. code Docs Qty Price
STING (V155M isoform; constitutively active) knockin NF-kB-SEAP and IRF-Lucia Reporter Monocytes
3-7 x 10e6 cells
thpd-m155
+-
$1,510.00

June, 18th 2018 - CONTENTS UPDATE NOTIFICATION
Please note that, for your convenience, these cells are now provided with QUANTI-Blue™ Solution.

NEW PRODUCT

STING (V155M isoform; constitutively active) knockin NF-κB-SEAP and IRF-Lucia Reporter Monocytes

THP1-Dual™ KI-hSTING-M155 cells were generated from THP1-Dual™ KO-STING cells by knockin of the intronless coding sequence (from the ATG to the TGA) of the M155 hSTING variant.

The allele M155 contains a unique point mutation (V155M) in the "wild-type" R232-RGR hSTING variant which confers gain-of-function and constitutive activation of STING [1-3]

This mutation is associated with an upregulation of IFN production and a chronic autoinflammatory disease, known as STING-associated vasculopathy with onset in infancy (SAVI) [2-3].

THP1‑Dual™ KI-hSTING-M155 cells are resistant to blasticidin and Zeocin™.

 

1. Yi G. et al., 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLOS One. 8:e77846.
2. Liu Y. et al., 2014. Activated STING in a vascular and pulmonary syndrome. N Engl J Med. 371:507-18.
3. Jeremiah N. et al., 2013. Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations. J Clin Invest. 124:5516-20.
 

Evaluation of IRF- and NF-κB-induced responses
Evaluation of IRF- and NF-κB-induced responses

THP1-Dual™ KI-hSTING-M155 cells were stimulated with 10 μg/ml of various cyclic dinucleotides (2’3’-cGAMP, 3’3’-cGAMP, 2’3’ c-di-AM(PS)2(Rp/Rp), cAIMP or cAIMP(PS)2Difluor(Rp/Sp), 30 μM BX795, 10 μM Brefeldin A, or 300 μg/ml Amlexanox. After overnight incubation, the IRF (panel a) and NF-κB(panel b) responses were determined using QUANTI‑Luc™ and QUANTI-Blue™ respectively. Bars represent the % activity on the signal in non-induced (NI) cells.

Evaluation of inhibitors on constitutive IRF-induced response
Evaluation of inhibitors on constitutive IRF-induced response

Dose inhibition of constitutively activated THP1-Dual™ KI-hSTING-M155 reporter cellswith Amlexanox, Brefeldin A and BX795 inhibitors. Starting concentrations of Amlexanox (300 μg/ml), Brefeldin A (10 μM), or BX795 (30 μM) were diluted in 3-fold series. After overnight incubation, the ISG response was determined using QUANTI‑Luc™. Data represent the % activity on the signal in non-induced (NI) cells without inhibitors.

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Specifications

Antibiotic resistance: Zeocin™, blasticidin

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 μg/ml)

Quality control:

  • The biallelic knockin (KI) of the human STING variant (M155) has been verified by functional assays, PCR and sequencing.
  • Reporter activity has been validated by stimulating the cells with STING ligands, such as c-di-AMP, cGAMP.
  • The cell line stability for 20 passages following thawing has been verified.
  • The cell line is 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™ KI-hSTING-M155 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™ (Lucia luciferase detection medium)
  • 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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Background

STING (stimulator of interferon genes) is essential for the interferon (IFN) response to cytoplasmic foreign or self-DNA. It directly senses cyclic dinucleotides (CDNs), which are important messengers in bacteria and innate immune agonists in mammals [1]. Distinct variants of human STING (hSTING) that affect CDN recognition and signal transduction have been identified: 

• R232 (R71‑G230-R232-R293): the most prevalent in the human population (~60%). Referred as the “wild-type” or 232R-RGR allele [2].

• HAQ (H71A230-R232-Q293): contains three non-synonymous single nucleotide substitutions; R71H, G230A and R293Q. This allele, found in ~20% of the population, is less sensitive to CDNs than the “wild-type” allele [2].

• H232 (R71‑G230-H232-R293): the most commonly used hSTING variant in structural studies [2].

 S154: a gain-of-function mutation resulting in constitutive STING activation [3].

• M155: a gain-of-function mutation resulting in constitutive STING activation [3,4].

• A162: a synthetic mutation (S162A) that confers hSTING sensitivity to DMXAA, a tumor vascular disrupting agent in mice [5].

THP1-Dual™ KI-STING cells are a family of reporter cells allowing the study of STING variation by monitoring the activation of the transcription factors ISRE (IFN-stimulated response elements) and NF-kB. They were generated from THP1-Dual™ KO-STING cells, which derive from the human THP-1 monocytes, by stable biallelic knockout of the endogenous HAQ hSTING gene and stable integration of two inducible secreted reporter genes: Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase). The Lucia luciferase gene is under the control of an ISG54 (interferon-stimulated gene) minimal promoter in conjunction with five ISRE. The SEAP gene is driven by an IFN-β minimal promoter fused to five copies of the NF-kB response element. As a result, they allow the simultaneous study of the IFN regulatory factor (IRF) and NF-kB pathways. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Luc, a Lucia™ detection reagent, and QUANTI-Blue, a SEAP detection reagent.

 

1. Sun L. et al., 2013. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 339:786-91.
2. Yi G. et al., 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLOS One. 8:e77846.
3. Liu Y. et al., 2014. Activated STING in a vascular and pulmonary syndrome. N Engl J Med. 371:507-18.
4. Jeremiah N. et al., 2013. Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations. J Clin Invest. 124:5516-20.
5. Gao P. et al., 2014. Binding-pocket and lid-region substitutions render human STING sensitive to the species-specific drug DMXAA. Cell Reports. 8:1668-76.

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