Ager Knockout RAW 264.7 Cell Line

The Ager Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited murine macrophage cell line with targeted disruption of the Ager gene, eliminating expression of the multi-ligand receptor RAGE. This loss-of-function model enables precise investigation of RAGE-mediated signaling without pharmacological interference. Through CRISPR/Cas9-mediated gene disruption, the Ager locus is rendered nonfunctional, providing an isogenic system for studying RAGE-dependent processes.

The parental RAW 264.7 cell line, derived from BALB/c mouse monocyte/macrophage-like cells, is extensively used to examine macrophage functions including phagocytosis, innate immunity, antigen presentation, and inflammatory cytokine production. These cells are especially valuable for studying LPS/TLR4 signaling and NF-??B activation, mimicking activated macrophage behavior. The Ager knockout derivative maintains these characteristics while permitting dissection of RAGE-specific contributions.

RAGE is a pattern recognition receptor recognizing AGEs, HMGB1, S100A8/A9, S100B, and amyloid-??. Ligand binding recruits mDia1, activating Rho GTPases and downstream kinases. Key pathways include NF-??B (via IKK), MAPK cascades (ERK1/2, p38, JNK) leading to AP-1, PI3K/Akt/mTOR, and JAK/STAT3. RAGE-induced NADPH oxidase generates ROS. Transcriptional targets include TNF-??, IL-6, VCAM-1, ICAM-1, MMP-9, and VEGF. RAGE functionally interacts with TLR2/4 via adaptors MyD88 and TIRAP, amplifying inflammation. In macrophages, RAGE signaling promotes sustained cytokine secretion and oxidative stress.

In RAW 264.7 macrophages, RAGE orchestrates prolonged NF-??B activation and cytokine release upon stimulation with DAMPs. Deletion of Ager removes this signaling arm, enabling clear differentiation of RAGE-dependent versus -independent pathways. The model is ideal for investigating RAGE?CTLR4 crosstalk, given the cells?? robust LPS responses. It also facilitates studies on macrophage migration and phagocytosis in contexts such as diabetic complications, atherosclerosis, and neuroinflammation.

Research applications include mechanistic analysis of RAGE-driven NF-??B and MAPK signaling, screening of RAGE inhibitors, and functional studies in inflammation, cancer, and ischemia-reperfusion injury. The line supports assays such as ELISA for cytokine secretion, NF-??B luciferase reporters, cell migration and invasion assays, co-immunoprecipitation of mDia1, and flow cytometry for RAGE surface expression. Phospho-protein analysis and ROS detection further elucidate oxidative and inflammatory pathways. The Ager Knockout RAW 264.7 Cell Line is a precise tool for both basic and translational research. For further information, please contact Ascent Research.