THP1-Dual™ KO-cGAS Cells

Notification: Reference #thpd-kocgas-av can only be ordered together with reference #thpd-kocgas.

cGAS knockout NF-κB-SEAP and IRF-Lucia luciferase reporter monocytes

THP1-Dual KO-cGAS pathway

THP1-Dual^™ KO-cGAS cells were generated from THP1-Dual^™ cells by stable knockout of the cGAS gene. They derive from human THP-1 monocytes, a cell line often used to study DNA sensing pathways as they express all the cytosolic DNA sensors identified so far (with the exception of DAI).

THP1-Dual^™ and THP1-Dual^™ KO-cGAS cells stably express two inducible secreted reporter genes: Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase). The Lucia luciferase reporter gene is under the control of an ISG54 (interferon-stimulated gene) minimal promoter in conjunction with five IFN-stimulated response elements. 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 the NF-kB pathway by assessing the activity of Lucia luciferase and SEAP, respectively.  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.

THP1-Dual™ KO-cGAS and THP1-Dual™ cells can be used to study the role of cGAS by monitoring IRF-induced Lucia luciferase activity. THP1-Dual™ KO-cGAS cells are resistant to blasticidin and Zeocin®.

References:

1. Sun L. et al., 2013. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339(6121):786-91.
2. Gao P. et al., 2013. Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell. 153(5):1094-107.
3. Ablasser A. et al., 2013. cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING. Nature. 498(7454):380-4.

Figures

Validation of cGAS knockout by Western blot (Wes™)

Analysis of lysates from the THP1-Dual™ (WT) and THP1-Dual™ KO-cGAS (KO) cells using Anti-cGAS, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the human cGAS protein (59 KDa).

IRF INDUCTION (Lucia luciferase reporter)

Stimulation of THP1-Dual™ (parental cell line) and THP1-Dual™ KO-cGAS cells with VACV70/LyoVec™ (1 μg/ml), poly(dA:dT)/LyoVec™ (100 ng/ml), and 2’3’-cGAMP (1 μg/ml). Human IFN-α (1x10⁴ U/ml) and IFN-β (1x10⁴ U/ml) serve as positive controls. Human TNF-α (10 ng/ml) has been included as a negative control. After a 24h incubation, IRF activation 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%).

NF-kB INDUCTION (SEAP reporter)

THP1-Dual™ and THP1-Dual™ KO-cGAS cells were incubated with TNF-α (0.1 ng/ml), Pam3CSK4 (0.1 ng/ml; TLR1/2 ligand), LPS-EB Ultrapure (100 ng/ml; TLR4 ligand), VACV70/LyoVec™ (1 μg/ml), poly(dA:dT)/LyoVec™ (100 ng/ml), 2’3’-cGAMP (3 μg/ml) and hIFN-α (1x10⁴ U/ml). After a 24h incubation, NF-kB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 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
Biallelic cGAS knockout is verified by functional assays, PCR, and DNA sequencing.
These cells are guaranteed mycoplasma-free.

Contents

• 1 vial of THP1-Dual™ KO-cGAS 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 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)

Details

THP1 reporter cells are a family of cells derived from the human monocytic THP-1 cell line, which naturally expresses many pathogen recognition receptors (PRRs), including Toll-like receptors.

They respond to ligands for certain TLRs; namely, TLR2, TLR1/2, TLR2/6, TLR4, TLR5 and TLR8. These cells can also be used to study DNA sensing pathways, as they are highly responsive to PRR agonists that trigger interferon (IFN) signaling pathways.

THP1‑Dual™ cells feature two reporter genes that enable the simultaneous study of the NF-κB and IFN signaling pathways.

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) is a critical cytosolic DNA sensor that triggers innate immune responses through the production of type I interferons (IFNs) [1]. In response to cytosolic double‑stranded DNA (dsDNA), cGAS produces the cyclic dinucleotide (CDN) 2’3’-cGAMP. CDNs bind directly to STING, leading to TBK1‑IRF3-mediated activation of IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG). The most potent agonist of human STING is 2’3’-cGAMP [2,3].

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