GNPNAT1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population generated from the HAP1 human cell line. This product features targeted disruption of the GNPNAT1 gene, which encodes glucosamine-6-phosphate N-acetyltransferase 1, a key enzyme in the hexosamine biosynthetic pathway. The polyclonal format comprises a heterogeneous pool of edited cells, providing a robust loss-of-function model without clonal artifacts. This population is suitable for functional genomic screens and mechanistic studies in a near-haploid genetic background.
HAP1 cells are a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia line. Their haploid genome simplifies genetic analysis, making them ideal for forward genetic screens, functional validation, and studying recessive phenotypes. These cells retain features of hematopoietic progenitors yet exhibit adherent growth and stable karyotype, facilitating routine culture and high-throughput applications. The near-haploid state enhances the penetrance of gene disruptions, enabling clearer loss-of-function readouts, particularly advantageous for dissecting metabolic pathways like hexosamine biosynthesis where redundancy can mask phenotypes.
GNPNAT1 catalyzes the acetylation of D-glucosamine-6-phosphate to N-acetyl-D-glucosamine-6-phosphate, a rate-limiting step in the hexosamine biosynthetic pathway that produces UDP-N-acetylglucosamine (UDP-GlcNAc). It functions downstream of GFPT1 and GFPT2, which sense glucose, glutamine, and insulin signals. GNPNAT1 cooperates with PGM3 and UAP1 to generate UDP-GlcNAc, the essential donor for N-linked glycosylation and for O-GlcNAc modification of proteins by O-GlcNAc transferase (OGT). OGT and the glycosidase MGEA5 dynamically regulate O-GlcNAcylation, linking nutrient status to protein function in processes such as signal transduction, stress responses, and transcription. Thus, GNPNAT1 is a central regulator of glycosylation, impacting cell adhesion, signaling, and proliferation.
In the HAP1 background, disruption of GNPNAT1 provides a clean system to interrogate hexosamine-dependent glycosylation without genetic redundancy. The near-haploid genome ensures uniform loss of function, facilitating quantitative analysis of O-GlcNAc dynamics, N-glycan profiles, and metabolic fluxes. Originating from chronic myeloid leukemia, this model is particularly relevant for studying how aberrant hexosamine pathway activity and O-GlcNAcylation drive cancer cell survival, proliferation, and therapy resistance. It also enables modeling of congenital disorders of glycosylation linked to GNPNAT1 deficiency, bridging metabolic research with translational insights.
This knockout pool is compatible with diverse functional assays: western blotting for O-GlcNAc and OGT levels, mass spectrometry of N-glycans, RT-qPCR of pathway genes (GFPT1, PGM3, UAP1), and metabolic labeling with azido sugars. Phenotypic screens can explore cell proliferation and apoptosis under altered glucose or glutamine supply, complemented by immunofluorescence for glycoprotein localization. Applications include investigations into metabolic reprogramming in leukemia, O-GlcNAc-mediated signaling, and glycosylation disorder pathogenesis. For further information or custom applications, contact Ascent Research.
