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THP1-Dual™ KO-SAMHD1 Cells

THP1-Dual™ KO-SAMHD1 Cells Unit size Cat. code Docs Qty Price
Human THP-1 Monocytes - SAMHD1 knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
thpd-kosamhd1
+-
$1,568.00

SAMHD1 knockout NF-κB-SEAP and IRF-Lucia luciferase reporter monocytes

Signaling pathways in THP1-Dual™ KO-SAMHD1 Cells
Signaling pathways in THP1-Dual™ KO-SAMHD1 Cells

THP1-Dual™ KO-SAMHD1 cells were generated from THP1-Dual™ cells through the stable biallelic knockout of the SAMHD1 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-SAMHD1 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.

SAMHD1 (sterile alpha motif and histidine-aspartate domain-containing protein 1) is a predominantly nuclear enzyme playing a major role in nucleotide homeostasis by balancing cellular dNTP (deoxynucleoside triphosphate) levels [1]. Furthermore, it can influence viral activity and act as an important negative regulatory factor of the human innate immune response [1-3]. 

More details More details

 

Key features:

  • Biallelic knockout of the SAMHD1 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 SAMHD1 signaling pathways
  • Screening of interactions between SAMHD1 and other signaling protein
  • Study the role of SAMHD1 in innate immunity, tumorigenesis, or viral replication

 

1. Deutschmann et al., 2021. SAMHD1 … and Viral Ways around It. Viruses. 2021;13(3):395.
2. Rice et al., 2009. Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as a regulator of the innate immune response. Nature genetics, 41(7), 829–832.
3. Oo et al., 2022. Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication. jbc.2022.101635

Figures

Validation of SAMHD1 knockout
Validation of SAMHD1 knockout

Figure 1: Validation of SAMHD1 KO. (A) The targeted SAMHD1 region in THP1-Dual™ (WT; blue arrow) parental cells and THP1‑Dual™ KO-SAMHD1 (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-SAMHD1 cells were generated by a biallelic deletion of 132 bp in exon 7, causing the inactivation of SAMHD1. The WT PCR product is 868 bp, whereas the truncated KO band measures only 736 bp. 
(B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-SAMHD1 (KO) cells were analyzed using an anti-human SAMHD1 antibody, followed by an HRP‑conjugated anti‑rabbit secondary antibody (JESS™ system). As expected a band was detected at ~70 kDa in the WT cells only (green arrow). 

Functional validation of SAMHD1 knockout (NF-κB response)
Functional validation of SAMHD1 knockout (NF-κB response)

Figure 2: NF-κB responses in THP1-Dual™ -derived cells. THP1-Dual™ and THP1 Dual™ KO-SAMHD1 cells were incubated with 1 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 1000 U/ml hIFN-β (IRF-Lucia positive control), 100 ng/ml LPS-EK Ultrapure and 100 µg/ml LPS-SM Ultrapure (both TLR4 agonists), 10 µg/ml R848 (TLR7/8 agonist), 1 µg/ml VacV70 complexed with LyoVec™ (CDS agonist), 1 µg/ml 3p-hpRNA complexed with LyoVec™ (RIG-I agonist) and 30 µg/ml, 2’3’-cGAMP (STING agonist). 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).

Functional validation of SAMHD1 knockout (IRF response)
Functional validation of SAMHD1 knockout (IRF response)

Figure 3: IRF responses in THP1-Dual™ -derived cells. THP1-Dual™ and THP1-Dual™ KO-SAMHD1 cells were incubated with 1 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 100 ng/ml LPS-EK Ultrapure and 1 µg ml LPS-SM Ultrapure (both TLR4 agonists), 10 µg/ml R848 (TLR7/8 agonist), 1 µg/ml VacV70 complexed with LyoVec™ (CDS agonist), 1 µg/ml 3p-hpRNA complexed with LyoVec™ (RIG-I agonist) and 30 µg/ml 2’3’-cGAMP (STING agonist). 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).

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Specifications

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 U/ml Penicillin, 100 μg/ml Streptomycin, 100 μg/ml Normocin™

Antibiotic resistance: Zeocin®Blasticidin

Quality control: 

  • Biallelic SAMHD1 gene knockout has been verified by PCR, western blot, DNA sequencing, and functional assays.
  • The stability of this cell line for 20 passages following thawing has been verified.
  • THP1-Dual™ KO-SAMHD1 cells are guaranteed mycoplasma-free.

This product is covered by a Limited Use License (See Terms and Conditions).

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Contents

  • 1 vial of THP1-Dual™ KO-SAMHD1 cells (3-7 x 106 cells)  in a cryovial or shipping flask
  • 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 pouch of QUANTI-Luc™
  • 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

Dry Ice Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

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Details

SAMHD1 plays a variety of roles in cell biology,  immunology, and virology [1].

Using its phosphohydrolase activity, SAMHD1 converts dNTPs into inorganic triphosphates (PPPis) and deoxynucleosides (dN) to ensure efficient genome replication in dividing cells [2]. Moreover, it facilitates the replication fork progression and prevents DNA damage by actively recruiting members of the DSB (double-strand break) repair machinery [1].

Additionally, SAMHD1 can interfere with mediators of NF-κB and IRF signaling pathways, thus preventing an excessive antiviral and proinflammatory response [2]. Depending on whether a virus benefits from these suppressive effects on the host immune system, SAMHD1 allows for either antiviral or proviral events. SAMHD1 has been reported to limit HIV-1 replication while promoting Zika or SARS-CoV-2 infection [3].

Defects or alterations in the SAMHD1 gene have been associated with tumor development and Aicardi-Goutières syndrome, an autoimmune disorder characterized by spontaneous hyperactivation of the type I IFN pathway and excessive IFN-α production. This syndrome is also induced by mutations in other nuclease-coding genes such as TREX1 or RNASEH2 [3,4]. 

 

1. Coggins et al., 2020. SAMHD1 Functions and Human Diseases. Viruses, 12(4), 382.
2. Deutschmann et al., 2021. SAMHD1 … and Viral Ways around It. Viruses.13(3):395.
3. Oo et al., 2022. Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication. jbc.2022.101635.
4. Rice et al., 2009. Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as a regulator of the innate immune response. Nature genetics, 41(7), 829–832.

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