RAW-Lucia™ ISG-KO-IRF5 Cells
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Murine RAW 264.7 macrophages - IRF5 knockout and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
IRF5 knockout reporter macrophages
RAW-Lucia™ ISG-KO-IRF5 cells were generated from the RAW-Lucia™ ISG cell line, which is derived from the RAW 264.7 murine macrophages, through the stable knockout of IRF5 gene.
IRF5 (interferon regulatory factor 5) is a critical transcription factor involved in the induction of type I interferon (IFN) downstream of pattern recognition receptors (PRRs), including the cytosolic DNA/cyclic dinucleotide sensors cGAS/STING, the cytoplasmic RNA sensors, RIG-I and MDA5, Toll-like receptors (TLRs), the NOD-like receptor NOD2, and C type lectin receptors such as Dectin 1 and Dectin 2 . Several lines of evidence indicate that, depending on the PRR that is triggered, IRF5 is activated through different mechanisms [1,2].
RAW-Lucia™ ISG-KO-IRF5 and RAW-Lucia™ ISG cells feature a Lucia luciferase reporter gene under the control of an ISG54 promoter enhanced by multimeric ISREs. Thus, activation of the IRF pathway can be readily assessed by monitoring the activity of the secreted Lucia luciferase in the supernatant using the QUANTI-Luc™ detection reagent. As expected, the IRF response is strongly diminished upon incubation with STING agonists, such as 2’3’-cGAMP. However, differential responses are observed when using DNA- or RNA-based agonists with varying transfection reagents. RAW-Lucia™ ISG-KO-IRF5 cells retain the full ability to respond to type I interferons (IFN-α and IFN-β) and lipopolysaccharide (a TLR4 agonist). These cells are resistant to Zeocin™.
Features of RAW-LUCIA™ ISG KO-IRF5 cells:
- Biallelic knockout of the IRF5 gene
- Readily assessable Lucia luciferase activity
Applications for RAW-LUCIA™ ISG KO-IRF5 cells:
- Defining the role of IRF5 in PRR-induced signaling
- Highlighting possible overlapping PRR activation or regulatory mechanisms (see 'Details' tab)
Validation of RAW-Lucia™ ISG KO-IRF5 cells:
- IRF5 knockout verified by PCR, DNA sequencing, and Western blot
- Functionally tested
1. Zhao G-N. et al., 2015. Interferon regulatory factors: at the crossroads of immunity, metabolism, and disease. Biochim Biophys Acta. 2015 Feb;1852(2):365-78.
2. Jefferies C.A., 2019. Regulating IRFs in IFN driven disease. Frontiers in immunology. Vol 10. Article 325.
Figure 1: Amplification of the targeted IRF5 region in RAW-Lucia™ ISG (WT) and RAW-Lucia™ ISG-KO-IRF5 (KO) cells. RAW-Lucia™ ISG-KO-IRF5 cells feature a biallelic deletion of 2173-base pairs (arrow).
Figure 2: Analysis of lysates from the RAW-Lucia™ ISG (WT) and RAW-Lucia™ ISG-KO-IRF5 (KO) cells using Anti-IRF5, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the IRF5 protein (56 kDa).
Figure 3: 2x105 RAW-Lucia™ ISG and RAW-Lucia™ ISG-KO-IRF5 cells were incubated with 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using the QUANTI-Luc™ detection reagent. Activity fold increase over non-induced cells is shown.
Figure 4: 2x105 RAW-Lucia™ ISG or RAW-Lucia™ ISG-KO-IRF5 cells were transfected with 1 μg/ml G3-YSD, 1 μg/ml VACV-70 (A, B), 1 μg/ml 3p-hpRNA, or 1 μg/ml 5’ppp-dsRNA (C, D) complexed with Lyovec™ (A, C) or LTX (B, D). After overnight incubation, the IRF response was assessed by measuring the bioluminescent activity of the Lucia luciferase in the supernatant using QUANTI-Luc™. Activity fold increase over non-transfected cells is shown.
Figure 5: 2x105 RAW-Lucia™ ISG or RAW-Lucia™ ISG-KO-IRF5 cells were incubated with 104 U/ml mouse IFN-α (mIFN-α), 104 U/ml mouse IFN-β (mIFN-β), or 1 μg/ml LPS EK Ultrapure (UP). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using the QUANTI-Luc™ detection reagent. Activity fold increase over non-induced cells is shown.
Antibiotic resistance: Zeocin™
Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™
- Biallelic IRF5 knockout has been verified by PCR, DNA sequencing, Western blot, and functional assays.
- The stability for 20 passages, following thawing, has been verified.
- These cells are guaranteed mycoplasma-free.
This product is covered by a Limited Use License (See Terms and Conditions).Back to the top
- 1 vial of RAW-Lucia™ ISG-KO-IRF5 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 mycoplasma, bacteria, and fungi.
- 1 ml of Zeocin™ (100 mg/ml)
- 1 pouch of QUANTI-Luc™
Shipped on dry ice (Europe, USA & Canada)Back to the top
RAW-Lucia™ ISG-KO-IRF5 cells have been functionally tested using various sources of nucleic acids (see the validation data document).
Several lines of evidence indicate that, depending on the PRR that is triggered, IRF5 is activated through different mechanisms. For example, nucleic acid sensing by RIG-I, MDA5, cGAS or STING, elicit IRF5 activation through TBK1 (TANK-binding kinase 1). IRF5 can also be activated by the IKKβ kinase downstream of the MAVS adaptor associated with RIG-I or MDA5, or downstream of the MyD88 adaptor associated with TLRs.As expected, the IRF response is strongly diminished when the RAW-Lucia™ ISG-KO-IRF5 cells are incubated with STING agonists, such as 2’3’-cGAMP. The IRF response is not completely abrogated because other IRFs, such as IRF3 have overlapping functions downstream in the STING signaling pathway. However, differential responses are observed when using DNA- or RNA-based agonists with varying transfection reagents. Such observations could highlight overlapping nucleic acid-sensing or regulatory mechanisms. RAW-Lucia™ ISG-KO-IRF5 cells retain a full ability to respond to type I interferons (IFN-α and IFN-β) and lipopolysaccharide (LPS).Back to the top