THP1-Dual™ KO-SAMHD1 Cells

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

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

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

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 causing the inactivation of SAMHD1. 
(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). 

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).

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).

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.

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 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

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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