HEK-Blue™ ISG-KO-STING Cells

STING Knockout IRF Reporter Cell Line

HEK-Blue™ ISG-KO-STING cells were specifically designed to study the STING/TBK1/IRF3 signaling pathway. HEK-Blue™ ISG-KO-STING cells were generated from the HEK-Blue™ ISG cells through the stable knockout of the STING gene.

The parental HEK-Blue™ ISG cells were derived from the PEAKrapid cell line (similar to ATCC® CRL-2828™) which itself was derived from the HEK293 cell line. Unlike their parental cell line, HEK-Blue™ ISG-KO-STING cells do not respond to cytosolic DNA, CDNs, and xanthenone derivatives, such as DMXAA.

HEK-Blue™ ISG-KO-STING cells can detect bioactive type I IFNs through the activation of the JAK-STAT-IRF9 pathway.

HEK-Blue™ ISG-KO-STING cells express a secreted embryonic alkaline phosphatase (SEAP) under the control of the IRF-inducible promoter comprised of five IFN-stimulated response elements (ISRE) fused to an ISG54 minimal promoter.

Levels of SEAP in the supernatant can be easily determined with QUANTI- Blue™, a reagent that turns purple/blue in the presence of SEAP and by reading the OD at 620-655 nm.

References:

1. Ishikawa H. & Barber G., 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678.
2. Burdette DL. et al., 2011. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478(7370):515-8.
3. ;Wu J. et al., 2013. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339(6121):826-30.

Figures

Response of HEK-Blue™ ISG and HEK-Blue™ ISG KO-STING cells to various CDNs, cytosolic dsDNA and IFN-β.
HEK-Blue™ ISG KO-STING and HEK-Blue™ ISG cells (wild-type cell line) were stimulated with 1x10³ U/ml human IFN-β, 1 μg/ml poly(dA:dT)/LyoVec™, 30 μg/ml 2’3’-cGAMP, 3’3’-cGAMP, 3’3’-cGAMP Fluorinated, 2’3’-c-di-AMP, cAIMP, and 2’3’-c-di-AM(PS)₂ (Rp,Rp). After 24h incubation, IRF activation was determined using QUANTI‑Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. The IRF induction of each ligand is expressed as % activity relative to that of human IFN-β at 1x10³ U/ml (taken as 100%).

Response of HEK-Blue™ ISG and HEK-Blue™ ISG KO-STING cells to various CDNs, cytosolic dsDNA and IFN-β.
The difference in activity between the two cell lines is expressed as the ratio WT/KO, which was obtained by dividing each value in figure 1 for HEK‑Blue™ ISG (WT) cells by the corresponding value for HEK-Blue™ ISG KO-STING cells.

Validation of STING knockout by Western blot (Wes™). Analysis of lysates from the HEK-Blue™ ISG (WT) and HEK-Blue™ ISG-KO-STING (KO) cells using Anti-STING, followed by an HRP-conjugated anti-mouse secondary antibody. The arrow indicates the expected band for the human STING protein (42 KDa).

Specifications

Antibiotic resistance: G418, Zeocin^®

Quality control: STING knockout is verified by functional assays (see validation sheet) and DNA sequencing. These cells are guaranteed mycoplasma-free.

Growth Medium: DMEM, 4.5 g/l glucose, 10% (v/v) fetal bovine serum (FBS), Pen-Strep (100 U/ml - 100 µg/ml), 100 µg/ml Normocin™, 2 mM L-glutamine

Test Medium: DMEM, 4.5 g/l glucose, 10% (v/v) heat-inactivated FBS (30 min at 56°C), Pen-Strep (100 U/ml - 100 µg/ml), 100 µg/ml Normocin™, 2 mM L-glutamine

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Zeocin^® (100 mg/ml)
• 1 ml Normocin™ (50 mg/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)

Details

STING (stimulator of interferon genes; also known as TMEM173, MITA, MPYS and ERIS) is essential for the interferon (IFN) response to cytosolic nucleic acids, such as microbial or self-DNA [1], and acts as a direct sensor of cyclic dinucleotides (CDNs) [2].

CDNs are important second messenger molecules in bacteria, affecting numerous responses of the prokaryotic cell. In mammalian cells, CDNs act as agonists of the innate immune response [3]. CDNs bind directly to and activate STING leading to a potent type I interferon (IFN) response.

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