The Dapk1 Knockout RAW 264.7 Polyclonal Cells product from Ascent Research provides a ready-to-use population of CRISPR/Cas9-edited cells in which the Dapk1 gene has been disrupted. This polyclonal knockout cell pool is derived from the widely used RAW 264.7 mouse macrophage cell line and is designed for researchers investigating DAPK1-mediated signaling, apoptosis, autophagy, and tumor suppression mechanisms. The gene disruption was achieved using CRISPR/Cas9 technology, resulting in a heterogeneous population of cells with targeted Dapk1 loss-of-function, suitable for diverse functional assays without the need for single-cell cloning.
The parental RAW 264.7 cell line was established from BALB/c mouse peritoneal macrophages transformed with Abelson murine leukemia virus. RAW 264.7 cells exhibit robust macrophage characteristics, including adherent growth, active phagocytosis, and the ability to mount inflammatory responses when stimulated with lipopolysaccharide or cytokines such as interferon-gamma. These features make RAW 264.7 an ideal host line for studying innate immunity, host-pathogen interactions, and the molecular regulation of macrophage effector functions.
DAPK1 is a calcium/calmodulin-dependent serine/threonine kinase that functions as a key tumor suppressor and regulator of programmed cell death. It is activated by calcium/calmodulin binding and by upstream signals from death receptors such as Fas ligand and TNF-??, as well as by ceramide, TGF-??, and oxidative stress. Upon activation, DAPK1 phosphorylates downstream targets including myosin light chain (MLC) to induce membrane blebbing and Beclin-1 to promote autophagy. DAPK1 also interfaces with the p53 pathway through p19ARF/MDM2, modulates mTOR signaling via TSC2, and interacts with proteins such as ERK1/2, 14-3-3, Pin1, and ZIP kinase. These interactions place DAPK1 at the crossroads of apoptosis, autophagy, and cell adhesion pathways, influencing processes such as anoikis, inflammation, and neuronal cell death.
In the RAW 264.7 macrophage context, Dapk1 disruption offers a valuable model to dissect the kinase??s roles in immune cell function. Macrophages are central to innate immunity and inflammation, and DAPK1 has been implicated in macrophage apoptosis, efferocytosis, and the balance between survival and death during immune challenge. This knockout cell population enables precise analysis of DAPK1-dependent signaling in response to classical macrophage stimuli, including LPS and IFN-??, and allows researchers to evaluate how loss of DAPK1 affects phagocytic capacity, cytokine production, and autophagic flux. Moreover, given DAPK1??s involvement in neurodegenerative diseases and cancer, these cells serve as a platform for studying macrophage-driven pathologies in vitro.
Typical applications of the Dapk1 Knockout RAW 264.7 Polyclonal Cells include apoptosis assays using Annexin V/propidium iodide staining, phagocytosis measurements, cytokine profiling (e.g., TNF-??, IL-6) via ELISA, autophagy assessment by LC3 puncta quantification, and co-immunoprecipitation studies to map DAPK1 interactomes. These cells also support drug screening efforts for DAPK1 modulators and functional studies in tumor-associated macrophage biology, neuroinflammation, and inflammatory bowel disease. For detailed protocols, technical specifications, or further assistance, please contact Ascent Research.
