RAW-Lucia™ ISG-KO-RIG-I Cells

RIG-I Knockout IRF-Inducible Lucia luciferase reporter mouse macrophages

RAW-Lucia™ ISG-KO-RIG-I 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 RIG-I gene. RIG-I (retinoic-acid-inducible protein 1, also known as Ddx58) is a cytoplasmic RNA helicase, that senses double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, leading to production of type I interferons (IFNs) in infected cells.

RAW-Lucia™ ISG-KO-RIG-I 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-RIG-I and RAW-Lucia™ ISG cells can be used to study the role of RIG-I 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-RIG-I cells to murine type I IFNs is unaffected by the knockout of the RIG-I gene. As expected, the ability of RAW-Lucia™ ISG-KO-RIG-I cells to respond to transfected RNA, such as 5'ppp-dsRNA/LyoVec and poly(I:C) HMW/LyoVec, is greatly reduced.

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

Figures

Analysis of lysates from the RAW-Lucia™ (WT) and RAW-Lucia™ ISG-KO-RIG-I (KO) cells using Anti-RIG-I, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the murine RIG-I protein (102 KDa).

Response of RAW-Lucia™ ISG-KO-RIG-I cells to various stimuli. RAW-Lucia™ ISG-KO-RIG-I 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, 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 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: 
RIG-I 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)