RAW-Lucia™ ISG-KO-TBK1 Cells

TBK1 Knockout IRF-Inducible Lucia luciferase reporter mouse macrophages

RAW-Lucia™ ISG-KO-TBK1 cells were generated from the RAW-Lucia™ ISG cell line, which is derived from the murine RAW 264.7 macrophage cell line, through the stable knockout of the TBK1 gene. TBK1  (TANK-Binding Kinase 1) functions as a key node protein in several cell signaling pathways, including innate immune response, cell growth and proliferation.

RAW-Lucia™ ISG-KO-TBK1 and RAW-Lucia™ ISG cells express a secreted reporter gene, Lucia luciferase, under the control of the I-ISG54 promoter which is comprised of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

RAW-Lucia™ ISG-KO-TBK1 and RAW-Lucia™ ISG cells can be used to study the role of TBK1 by monitoring IRF-induced Lucia luciferase activity. The levels of IRF-induced Lucia in the cell culture supernatant can be easily monitored using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent.

The response of RAW‑Lucia™ ISG-KO-TBK1 cells to murine type I interferons (IFNs) is unaffected by the knockout of the TBK-1 gene. As expected, RAW‑Lucia™ ISG-KO-TBK1 cells do not respond to cyclic dinucleotides (e.g. 2'3'-cGAMP and c-di-AMP) and to transfected double-stranded DNA, such as poly(dA:dT)/LyoVec and VACV70/LyoVec.

RAW-Lucia™ ISG-KO-TBK1 cells are resistant to Zeocin®.

Figures

Analysis of lysates from the RAW-Lucia™ (WT) and RAW-Lucia™ ISG‑KO‑TBK1 (KO) cells using Anti‑TBK‑1, followed by an HRP‑conjugated anti‑rabbit secondary antibody. The arrow indicates the expected band for the murine TBK‑1 protein (83 kDa).

Response of RAW-Lucia™ ISG-KO-RIG-I cells to various stimuli. RAW-Lucia™ ISG-KO-TBK1 and RAW-Lucia™ ISG cells (parental cell line) were incubated with VACV70/LyoVec™ (1 µg/ml), poly(dA:dT)/LyoVec™ (1 µg/ml), poly(I:C) HMW/LyoVec™ (1 µg/ml), 5'ppp-dsRNA/LyoVec™ (1 µg/ml), 2'3'-cGAMP (3 µg/ml) and c-di-AMP (3 µg/ml). Mouse IFN-α (1x10⁴ U/ml) and IFN-β (1x10⁴ U/ml) serve as positive controls. Non-induced cells (NI) have been included as a negative control. After a 24h incubation, IRFactivation 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 mIFN-β at 1x10⁴ U/ml (taken as 100%).

Specifications

Antibiotic resistance: Zeocin®

Growth Medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™ supplemented with Zeocin®.

Quality Control: 
TBK1 knockout is verified by functional assays and DNA sequencing to confirm frameshift mutation/deletion.

The cells are guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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