cGAS KO Reporter RAW 264.7 Cells

cGAS Knockout IRF-Inducible Lucia luciferase reporter mouse macrophages

RAW-Lucia™ ISG-KO-cGAS 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 cGAS gene.
RAW-Lucia™ ISG-KO-cGAS 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 264.7 have been reported to express several CDSs, including cGAS [1]. RAW-Lucia™ ISG-KO-cGAS cells allow the monitoring of IRF activation by determining the activity of Lucia luciferase. 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.

RAW-Lucia™ ISG-KO-cGAS cells respond to interferons (e.g. IFN-α and IFN-β), cyclic dinucelotides (e.g. 2'3'-cGAMP and c-di-GMP) and transfected poly(I:C). However, as expected, they respond very poorly to transfected DNA, such as poly(dA:dT)/LyoVec and VACV70/LyoVec.

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

Reference:

1. Lam E. et al., 2014. Adenovirus Detection by the cGAS/STING/TBK1 DNA Sensing Cascade. J Virol. 88(2):974-81.

Figures

PCR amplification of the targeted region in the RAW-Lucia™ ISG-KO-cGAS (KO) and RAW-Lucia™ ISG (WT) cells. MWM = molecular weight marker

Analysis of lysates from the RAW-Lucia™ (WT) and RAW-Lucia™ ISG-KO-cGAS (KO) cells using Anti-cGAS, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the cGAS protein (59 KDa).

Stimulation of RAW-Lucia™ ISG-KO-cGAS and RAW-Lucia™ ISG cells (parental cell line) with poly(dA:dT)/LyoVec™ (1 µg/ml), VACV70/LyoVec™ (1 µg/ml), poly(I:C)/LyoVec™ (1 µg/ml), and 2'3'-cGAMP (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: 

cGAS 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)