Description

The Mettl3 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line that disrupts the Mettl3 gene in the well-characterized RAW 264.7 mouse macrophage background. This loss-of-function model enables precise investigation of N6-methyladenosine (m6A) RNA modification in macrophage biology by eliminating the catalytic subunit of the primary m6A methyltransferase complex. The resulting cell line serves as a critical tool for dissecting epitranscriptomic regulation of immune cell functions without the limitations of small-molecule inhibitors.

RAW 264.7 cells are a widely used murine macrophage line derived from an Abelson leukemia virus-induced tumor. They faithfully recapitulate key macrophage attributes, including robust phagocytic activity, the capacity to produce a broad spectrum of cytokines (e.g., TNF-??, IL-6, IL-1??), and antigen presentation. Their well-documented responsiveness to inflammatory challenges such as lipopolysaccharide (LPS) and interferon-?? makes them a preferred model for studying innate immunity, host-pathogen interactions, and inflammatory signaling. In the knockout context, this background allows direct examination of how m6A modifications shape macrophage effector functions.

METTL3 acts as the catalytic core of the METTL3-METTL14 heterodimeric methyltransferase complex, which co-transcriptionally deposits m6A marks on nascent mRNAs. Its expression is under the control of upstream transcription factors including MYC and SP1, and it is modulated by the PI3K/AKT and MAPK signaling cascades. Within the writer complex, METTL3 interacts with regulatory subunits such as WTAP, VIRMA, ZC3H13, and RBM15, which guide substrate selection and methylation efficiency. Key downstream targets like SOCS1, MYC, and LEF1 acquire m6A modifications that are decoded by YTH-domain readers (YTHDF1/2/3, YTHDC1) and can be erased by the demethylases FTO and ALKBH5. Through these interactions, METTL3 dynamically influences mRNA stability, splicing, nuclear export, and translation.

In the macrophage lineage, METTL3-mediated m6A methylation has been linked to the regulation of innate immune responses, including the control of cytokine synthesis, phagocytosis, and cell polarization. By ablating Mettl3 in RAW 264.7 cells, researchers can directly assess the impact of epitranscriptomic remodeling on processes such as LPS-induced inflammatory gene expression, macrophage activation states, and the cross-talk between metabolism and immunity. This knockout cell line thus provides a physiologically relevant platform for uncovering m6A-dependent regulatory nodes in macrophage function.

The Mettl3 Knockout RAW 264.7 Cell Line is suitable for a diverse range of experimental applications. Standard molecular techniques such as Western blotting and RT-qPCR enable confirmation of target gene disruption and quantification of downstream transcriptional changes. Epitranscriptomic profiling via m6A RNA immunoprecipitation coupled with high-throughput sequencing (MeRIP-seq) permits genome-wide mapping of methylation alterations. Functional readouts, including flow cytometry for surface marker analysis, multiplex cytokine profiling, and phagocytosis assays, provide comprehensive phenotypic characterization. This model is particularly valuable for researchers studying inflammation, cancer immunology, epitranscriptomic mechanisms, and the development of therapies targeting RNA modifications. For additional information or to acquire this knockout cell line, please contact Ascent Research.