THP1-Dual™ KO-TREX1 Cells

TREX1 knockout NF-κB-SEAP and IRF-Lucia luciferase reporter monocytes

Signaling pathways in THP1-Dual™ KO-TREX1 Cells

THP1-Dual™ KO-TREX1 cells were generated from THP1-Dual™ cells by stable biallelic knockout of the TREX1 gene. Human THP1 monocytes or derived macrophages are a common cellular model to study DNA sensing as they naturally express all cytosolic DNA sensors identified so far (except DAI). THP1-Dual™ KO-TREX1 cells feature two inducible reporter genes allowing the concomitant study of the IRF and NF-κB pathways, by monitoring the Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase) activities, respectively.

TREX1 (three prime repair exonuclease 1) is a major cellular 3'->5' exonuclease that plays a crucial role in maintaining immune homeostasis [1,2].

Key features:

• Biallelic knockout of the TREX1 gene
• Functionally validated with a selection of PRR ligands and cytokines
• Readily assessable Lucia luciferase and SEAP reporter activities

Applications:

• Study of IRF and NF-kB-dependent TREX1 signaling pathways
• Screening of interactions between TREX1 and other signaling protein
• Study the role of TREX1 in innate immunity

1. Kavanagh D. et al., 2008. New roles for the major human 3'-5' exonuclease TREX1 in human disease. Cell Cycle. 7(12):1718-25.
2. Hasan M. & Yan N., 2014. Safeguard against DNA sensing: the role of TREX1 in HIV-1 infection and autoimmune diseases. Front Microbiol. 5:193.

Figures

Figure 1: Validation of TREX1 KO. (A) The targeted TREX1 region in THP1-Dual™ (WT; blue arrow) parental cells and THP1‑Dual™ KO-TREX1 (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-TREX1 cells were generated by a biallelic deletion causing the inactivation of TREX1. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-TREX1 (KO) cells were analyzed using an anti-human TREX1 antibody, followed by an HRP‑conjugated anti-rabbit secondary antibody (Wes™ system). As expected a band was detected at ~33 kDa in the WT cells only (green arrow).

Figure 2: NF-κB responses in THP1-Dual™ -derived cells. THP1-Dual™ and THP1 Dual™ KO-TREX1 cells were incubated with 10 ng/ml human (h)TNF-α (NF κB-SEAP positive control), 10 000 U/ml hIFN-α, 10,000 U/ml hIFN-β (IRF-Lucia positive control), 1 μg/ml Poly(dA:dT)* and 1 μg/ml Poly(I:C) LMW* (both PRR agonists), 1 μg/ml VacV70*(CDS agonist), 1 μg/ml 5’ppp-dsRNA* (RIG-I agonist), 30 μg/ml 2’3’-cGAMP and 30 μg/ml c-di-AMP (both STING agonists). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI‑Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

* complexed with LyoVec™

Figure 3: IRF responses in THP1-Dual™ -derived cells. THP1-Dual™ and THP1-Dual™ KO-TREX1 cells were incubated with 1 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 10,000 U/ml hIFN-α, 1 μg/ml Poly(dA:dT)* and 1 μg/ml Poly(I:C) LMW* (both PRR agonists), 1 μg/ml VacV70*(CDS agonist), 1 μg/ml 5’ppp-dsRNA* (RIG-I agonist), 30 μg/ml 2’3’-cGAMP and 30 μg/ml c-di-AMP (both STING agonists). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI‑Luc™. The IRF induction of each ligand is expressed relative to that of hIFN-β at 1x10³ U/ml (mean ± SEM).

* complexed with LyoVec™

Specifications

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum,  Pen-Strep (100 U/ml-100 μg/ml), 100 μg/ml Normocin™.

Antibiotic resistance: Blasticidin,  Zeocin®

Quality control: 

• Biallelic TREX1 gene knockout has been verified by PCR, western blot, sequencing, and functional assays.
• The stability of this cell line for 20 passages following thawing has been verified.
• THP1-Dual™ KO-TREX1 cells are guaranteed mycoplasma-free.

Contents

• 1 vial of THP1-Dual™ KO-TREX1 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

TREX1 (also known as DNase III) is a major DNA-sensor nuclease in the cytoplasm.

The primary role of TREX1 is to target cellular DNA originating from aberrant replication and recombination [1]. TREX1 is bound to the ER (endoplasmic reticulum) and is able to degrade both single-stranded and double-stranded DNA as well as single-strand RNA. As a result, it blocks the activation of the cGAS-STING pathway, and thus dampening the nucleic acid sensor response. Therefore, it is thought to be a negative regulator of interferon (IFN) signaling preventing autoimmune diseases. Its acidic counterpart, DNase2, shares the same function, but is located in the lysosomes [2].

However,  TREX1 function also promotes protumor and -viral responses by degrading tumor- or virus-derived DNA that would otherwise stimulate the cGAS-STING pathway and elicit an immune response [2-3]. Unlike SAMHD1, another enzyme with nuclease activity, TREX1 boosts HIV-1 infection [4]. Mutations in TREX1 have been associated with a variety of disorders, such as the Aicardi–Goutières syndrome, Systemic Lupus Erythematosus or hereditary vascular retinopathy [1-4].

1. Kavanagh D. et al., 2008. New roles for the major human 3'-5' exonuclease TREX1 in human disease. Cell Cycle. 7(12):1718-25.
2. Baris, Adrian M et al., 2021. “Nucleic Acid Sensing in the Tumor Vasculature.” Cancers vol. 13,17 4452.
3. Hemphill et al., 2021. TREX1 as a Novel Immunotherapeutic Target. Front Immunol. Apr 1;12:660184.
4. Hasan M. & Yan N., 2014. Safeguard against DNA sensing: the role of TREX1 in HIV-1 infection and autoimmune diseases. Front Microbiol. 5:193 

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