The Anxa3 Knockout RAW 264.7 Polyclonal Cells product offers a CRISPR/Cas9-edited polyclonal knockout cell population derived from the murine macrophage cell line RAW 264.7, in which the Anxa3 gene has been disrupted to create a loss-of-function model. This polyclonal format provides a heterogeneous pool of edited cells, enabling robust and flexible analysis of gene function without the clonal variability associated with single-cell-derived lines. The knockout is generated through CRISPR/Cas9-mediated gene disruption, resulting in a versatile tool for studying the role of Anxa3 in macrophage biology. The product is supplied as a ready-to-use population, suitable for a wide range of downstream applications in immunology and cell biology research.
The host cell line, RAW 264.7, is a widely used mouse macrophage model established from BALB/c mice transformed with Abelson murine leukemia virus. These cells exhibit key characteristics of activated macrophages, including robust phagocytic activity, cytokine secretion, and responsiveness to inflammatory stimuli such as lipopolysaccharide (LPS). RAW 264.7 cells are a cornerstone in studies of innate immunity, particularly in examining mechanisms of phagocytosis, host-pathogen interactions, and signal transduction pathways mediating inflammation. Their well-characterized behavior and genetic tractability make them an ideal platform for generating targeted gene knockouts to dissect molecular functions in a physiologically relevant context.
Anxa3 encodes a calcium-dependent phospholipid-binding protein that localizes to phagosomal membranes and facilitates critical steps in phagosome maturation. It functions downstream of TLR4 and MyD88-mediated signaling, activated by upstream regulators such as LPS, tumor necrosis factor alpha (TNF-??), interleukin-1 beta (IL-1??), and calcium ionophores. Anxa3 interacts with actin, phospholipids, and calcium ions, and forms complexes with Rab7 and S100A8/A9 to promote phagosome-lysosome fusion. This action involves recruitment of LAMP1, activation of Rab7, and reorganization of the actin cytoskeleton, ultimately leading to acidification and degradation of phagosomal contents. Additionally, Anxa3 modulates NF-??B signaling through interactions within the PI3K/Akt pathway, influencing the production of pro-inflammatory cytokines such as TNF-??. Disruption of Anxa3 thereby impairs these coordinated membrane dynamics and downstream inflammatory responses.
In the context of RAW 264.7 macrophages, Anxa3 knockout profoundly impacts the cell’s ability to execute efficient phagocytosis and pathogen clearance, as well as to regulate inflammatory cytokine output. This model is highly relevant for investigating diseases driven by dysregulated macrophage function, including chronic inflammation, sepsis, cancer, and autoimmune disorders. By eliminating Anxa3 expression, researchers can dissect its specific contributions to phagosome maturation, NF-??B-mediated transcriptional responses, and cross-talk between calcium signaling and innate immune pathways. The polyclonal nature of the knockout population also allows for the assessment of phenotypic variability, providing a more comprehensive view of gene function than clonal isolates.
This polyclonal knockout cell product is suitable for a broad spectrum of experimental applications aimed at elucidating macrophage biology. Typical assays include western blotting to confirm Anxa3 loss, phagocytosis assays using fluorescent beads, immunofluorescence staining for lysosome-phagosome fusion markers such as LAMP1, and cytokine ELISAs to quantify TNF-?? secretion. Further functional analyses can involve RT-qPCR for Anxa3 mRNA, flow cytometry for surface receptor profiling, bacterial killing assays, calcium imaging, and co-immunoprecipitation to map interacting partners like Rab7 and actin. These tools enable investigations into phagocytosis mechanisms, macrophage-mediated inflammation, drug screening for immune modulators, host-pathogen interaction studies, and gene function analysis in innate immunity. For additional support or inquiries, please contact Ascent Research.
