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 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)

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™, 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.