THP1-Dual™ KO-IFI16 Cells

IFI16 knockout dual reporter monocytes

THP1-Dual™ KO-IFI16 cells were generated from THP1-Dual™ cells by stable knockout of the IFI16 (Interferon-γ-inducible protein 16) gene. IFI16 is an innate immune sensor with multiple functions; DNA sensor, regulator of interferon (IFN) expression, activator of the inflammasome, and regulator of genome function [1,2]. These cell lines were derived 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-IFI16 cells stably express two inducible secreted reporter genes: IFN-inducible Lucia luciferase and NF‑κB-inducible SEAP (secreted embryonic alkaline phosphatase). They can be used to study the role of IFI16 by monitoring IRF-induced Lucia luciferase activity, using QUANTI‑Luc™, a Lucia™ luciferase detection reagent.

THP1-Dual™ KO-IFI16 cells are resistant to the selectable markers blasticidin and Zeocin®.

References:

1. Jakobsen MR. & Paludan SR., 2014. IFI16: At the interphase between innate DNA sensing and genome regulation. Cytokine Growth Factor Rev. 25(6):649-55.
2. Unterholzner L. et al., 2010. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 11(11):997-1004.

Figures

Validation of IFI16 knockout. (A) The targeted IFI16 region in THP1-null (WT) and THP1-KO-IFI16 (KO) cells was amplified by PCR. THP1-KO-IFI16 cells feature a biallelic deletion. (B) Lysates from THP1-null (WT) and THP1-KO-IFI16 (KO) cells were analyzed by Western blot (Wes™) using an anti-human IFI16 antibody, followed by an HRP-conjugated anti-mouse secondary antibody.

IRF responses in THP1-Dual™ and THP1-Dual™ KO-IFI16 cells to various stimuli.
Cells were incubated with Poly(I:C) LMW/LyoVec™ (1 µg/ml), VACV-70/LyoVec™ (1 µg/ml), 2’3’-cGAMP (3 µg/ml), and c-di-AMP (10 µg/ml). Human IFN-α and IFN-β (1x10⁴ U/ml) serve as positive controls.
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 1x10⁴ U/ml (taken as 100%).

 

 

 

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 IFI16 knockout was verified by functional assays, PCR, and DNA sequencing.
These cells are guaranteed mycoplasma-free.

Contents

• 1 vial of THP1-Dual™ KO-IFI16 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)

Description

THP1-Dual™ and THP1-Dual™ KO-IFI16 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‑κB response element. As a result, they allow the simultaneous study of the IFN regulatory factor (IRF) pathway, by assessing the activity of Lucia luciferase and the NF-κB pathway, by monitoring the activity of SEAP. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI‑Luc™, a Lucia™ luciferase detection reagent and QUANTI-Blue™, a SEAP detection reagent.