Epas1 Knockout RAW 264.7 Cell Line

The Epas1 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the mouse macrophage RAW 264.7 line, designed to disrupt the Epas1 gene encoding hypoxia-inducible factor 2?? (HIF-2??). This gene-edited cell model provides a loss-of-function platform for studying hypoxia signaling in a monocyte/macrophage context. The cell line is generated through CRISPR/Cas9-mediated gene disruption, resulting in ablation of functional HIF-2?? protein, and is suitable for applications dissecting oxygen-sensing pathways and macrophage biology.

The RAW 264.7 cell line originates from BALB/c mice and was established by transformation with Abelson murine leukemia virus, yielding a widely used model of macrophage function. These cells exhibit robust phagocytic activity, cytokine production, and responsiveness to immune stimuli, making them a standard in vitro system for innate immunity and inflammation research. Their macrophage lineage provides a relevant background for investigating gene functions in monocyte/macrophage biology, particularly in pathological contexts such as tumor microenvironment and inflammatory diseases.

EPAS1 (HIF-2??) is a basic helix-loop-helix/PAS transcription factor that heterodimerizes with ARNT (HIF-1??) to bind hypoxia response elements (HREs) in target gene promoters. Under normoxic conditions, HIF-2?? is hydroxylated by prolyl hydroxylases (PHD2) and subsequently recognized by the von Hippel-Lindau tumor suppressor (pVHL), leading to ubiquitination and proteasomal degradation. Hypoxic stabilization of HIF-2?? enables transcriptional activation of genes such as VEGF, EPO, GLUT1, PDK1, and BNIP3, which orchestrate angiogenesis, erythropoiesis, metabolic reprogramming, and survival. HIF-2?? activity is also modulated by interacting cofactors including p300/CBP and factor-inhibiting HIF (FIH1), and is regulated upstream by mTOR signaling and cellular oxygen status.

In RAW 264.7 macrophages, HIF-2?? plays a critical role in mediating adaptive responses to hypoxia, including glycolytic shift, angiogenic factor secretion, and modulation of inflammatory cytokine profiles. Disruption of Epas1 in these cells impairs hypoxia-driven gene expression, providing a tool to elucidate HIF-2??-specific functions independent of HIF-1??. This model enables dissection of HIF-2?? contributions to macrophage polarization, tumor-associated macrophage phenotypes, and hypoxic inflammation, relevant to cancer, ischemia, and atherosclerosis research. By eliminating HIF-2??, researchers can assess its role in redox homeostasis and metabolic adaptation under low oxygen tension.

The Epas1 Knockout RAW 264.7 Cell Line is suited for multiple experimental workflows, including Western blotting to confirm HIF-2?? loss, RT-qPCR for downstream targets like Vegf and Glut1, ChIP-qPCR using anti-HIF-2?? antibodies, and HRE-luciferase reporter assays to measure transcriptional activity. It can be employed in drug screening for HIF-2?? inhibitors, metabolic flux analysis to evaluate glycolysis, and cytokine profiling under hypoxia to study immune modulation. Additionally, this line facilitates cell migration/invasion assays to probe macrophage behavior in ischemia models or tumor microenvironments. For further details or technical support, please contact Ascent Research.