RAW-Lucia™ ISG-KO-MDA5 Cells

MDA-5 knockout IRF-inducible Lucia luciferase reporter mouse macrophages

RAW-Lucia^™ ISG-KO-MDA5 cells were generated from RAW-Lucia^™ ISG cells through the stable knockout of the MDA-5 gene. These cells derive from the murine RAW 264.7 macrophage cell line, which has been reported to express many pattern recognition receptors (PRRs), including the dsRNA sensors MDA-5 [1] and RIG-I [2,3] along with their adaptor protein IPS-1 (also known as MAVS) [3].

RAW-Lucia^™ ISG-KO-MDA5 and RAW-Lucia^™ ISG cells can be used to study the role of MDA-5 in a mouse macrophage cell line by monitoring of interferon regulatory factor (IRF)-induced Lucia luciferase activity. They express the gene for secreted Lucia luciferase under the control of the I-ISG54 promoter which is comprised of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. 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-MDA5 cells are resistant to Zeocin®.

References:

1. Hasan M. et al., 2011. Antimicrobial peptides inhibit polyinosinic-polycytidylic acid-induced immune responses. J Immunol. 187(11):5653-9.
2. Melchjorsen J. et al., 2005. Activation of innate defense against a paramyxovirus is mediated by RIG-I and TLR7 and TLR8 in a cell-type-specific manner. J Virol. 79:12944-51.
3. Yamashita M. et al., 2013. Antiviral innate immunity disturbs podocyte cell function. J Innate Immun. 5:231-41.

Figures

PCR amplification of the targeted region in the RAW-Lucia™ ISG-KO-MDA5 (KO) and RAW-Lucia™ ISG (WT) cells. MWM = molecular weight marker

Analysis of lysates from the RAW-Lucia™ (WT) and RAW-Lucia™ ISG-KO-MDA5 (KO) cells using Anti-MDA-5, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the murine MDA-5 protein (117 KDa).

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: 
MDA-5 knockout is verified by PCR 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)