THP1-Dual™ KI-hSTING-S154 Cells [ NEW ]
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STING (N154S isoform; constitutively active) knockin NF-kB-SEAP and IRF-Lucia Reporter Monocytes
3-7 x 10e6 cells
STING (N154S isoform; constitutively active) knockin NF-κB-SEAP and IRF-Lucia Reporter Monocytes
THP1-Dual™ KI-hSTING-S154 cells were generated from THP1-Dual™ KO-STING cells by knockin of the intronless coding sequence (from the ATG to the TGA) of the S154 hSTING variant.
The allele S154 contains a unique point mutation (N154S) in the "wild-type" R232 hSTING variant [1,2].
This mutation confers gain-of-function and constitutive activation of STING with an upregulation of IFN production and is associated with a chronic autoinflammatory disease, known as STING-associated vasculopathy with onset in infancy (SAVI) .
THP1‑Dual™ KI-hSTING-S154 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.
THP1-Dual™ KI-hSTING-S154 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-BlueT™ respectively. Bars represent the % activity on the signal in non-induced (NI) cells.
Dose inhibition of constitutively activated THP1-Dual™ KI-hSTING-S154 reporter cells with 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.
Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/mlNormocin™, Pen-Strep (100 U/ml-100 μg/ml)
• The biallelic knockin (KI) of the human STING variant (S154) 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).Back to the top
- 1 vial of THP1-Dual™ KI-hSTING-S154 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.
IMPORTANT: Cells are shipped frozen. If cells are not frozen upon arrival, contact InvivoGen immediately.Back to the top
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 . 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 .
• HAQ (H71‑A230-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 .
• H232 (R71‑G230-H232-R293): the most commonly used hSTING variant in structural studies .
• S154: a gain-of-function mutation resulting in constitutive STING activation .
• 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 .
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.