THP1-Dual™ KI-hSTING-H232 Cells

STING (R232H) knockin NF-κB-SEAP and IRF-Lucia Reporter Monocytes

THP1-Dual™ KI-hSTING-H232 cells were generated from THP1-Dual™ KO-STING cells by knockin of the intronless coding sequence (from the ATG to TGA) of the R232H human STING variant. R71‑G230-H232-R293 has been identified as a natural variant allele of STING occurring in ~14% of the human population and is the most commonly used hSTING variant in structural studies [1]. This isoform is associated with a diminished response to bacterial and metazoan CDNs when compared to the prevalent R232 hSTING allele [1,2]. 

THP1‑Dual™ KI-hSTING-H232 cells are resistant to blasticidin and Zeocin®.

References:

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. Diner E. et al., 2013. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep 3(5):1355-61.

Figures

Response profile of THP1-Dual™ KI-hSTING-H232 and THP1-Dual™ KI-hSTING-R232 cells. Cells were stimulated with hIFN-β (1 x 10⁴ U/ml), DMXAA (100 μg/ml), 2’3’-cGAMP (30 μg/ml), 3’3’-cGAMP (30 μg/ml), 2’3’-cGAM(PS)2 (Rp/Sp) (30 μg/ml), 3’3’-cGAMP fluorinated (30 μg/ml), 2’3’-c-di-AMP (30 μg/ml), 3’3’-c-di-AMP (30 μg/ml), 2’3’-c-di-AM(PS)2 (Rp/Sp) (30 μg/ml) and TNF-α (10 ng/ml). A) After a 24h incubation, IRF induction was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI‑Luc™, a Lucia luciferase detection reagent. The IRF induction of each ligand is expressed relative to that of hIFN-β at 1 x 10⁴ U/ml (taken as 100%). B) After a 24h incubation, NF‑κB induction was determined using QUANTI‑Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 630 nm.

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 knockin (KI) of the human STING variant (H232) has been verified by functional assays, PCR and sequencing.
• Reporter activity has been validated using functional assays.
• 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).

Contents

• 1 vial of THP1-Dual™ KI-hSTING-S154 cells (3-7 x 10⁶ cells) in freezing medium
• 1 ml Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi
• 1 ml Zeocin™ (100 mg/ml)
• 1 ml Blasticidin (10 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)
• 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, and some areas in Asia)

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 (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 [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™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent, and QUANTI-Blue™ Solution, 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.