The EGLN3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population in which the EGLN3 gene has been disrupted to generate a loss-of-function model. This polyclonal knockout pool consists of HAP1 cells bearing heterogeneous genetic modifications at the target locus, providing a robust tool for studying EGLN3-dependent functions without the biases inherent in single-cell clones.
HAP1 is a human near-haploid suspension cell line derived from the KBM-7 chronic myeloid leukemia line, originally established from a male patient. Its near-haploid karyotype simplifies genetic manipulation and phenotypic analysis, making it a widely adopted host for CRISPR-based knockout screening and haploid genetic studies. HAP1 cells grow in suspension, facilitating large-scale culture and high-throughput applications.
EGLN3 encodes a prolyl hydroxylase that operates under normoxic conditions to hydroxylate hypoxia-inducible factor alpha subunits (HIF-1?? and HIF-2??) on conserved proline residues. This modification promotes recognition by the von Hippel?CLindau (VHL) E3 ubiquitin ligase complex, which includes Elongin B/C, CUL2, and RBX1, leading to ubiquitination and proteasomal degradation of HIF-??. In hypoxia, reduced oxygen availability inhibits EGLN3 activity, stabilizing HIF-?? and allowing its dimerization with HIF-1?? to activate transcription of target genes such as VEGF, GLUT1, and EPO. EGLN3 is regulated by HIF-1??, HIF-2??, TP53, and endoplasmic reticulum stress, and its downstream effects are mediated through HIF-??, NF-??B, and ATF4. The enzyme also interacts with OS-9, PKM2, and requires cofactors iron, 2-oxoglutarate, and oxygen, placing it at the center of the HIF-1 signaling pathway and cellular oxygen sensing.
Disruption of EGLN3 in HAP1 cells is expected to result in constitutive stabilization of HIF-?? subunits even under normoxic conditions, effectively mimicking a hypoxic cellular state. This polyclonal knockout model enables the dissection of HIF-dependent and HIF-independent roles of EGLN3 in a homogeneous genetic background that retains near-haploid genomic integrity. The use of a polyclonal population mitigates clonal adaptation artifacts and provides a more representative loss-of-function phenotype for high-throughput screening assays.
These cells are particularly suited for investigations into hypoxia signaling, cancer drug resistance, ischemic disease modeling, and metabolic adaptation. Experimental applications include western blotting for HIF-1?? accumulation, RT-qPCR analysis of HIF target gene induction (e.g., VEGF, GLUT1), and functional assays such as cell proliferation under hypoxia, migration, invasion, glucose uptake, and lactate production measurements. The polyclonal nature of the knockout pool also supports large-scale screens for hypoxia pathway modulators and drug sensitivity testing in normoxic versus hypoxic conditions. For more detailed product information and technical support, please contact Ascent Research.
