The KHK Knockout HAP1 Polyclonal Cells are a heterogeneous pool of HAP1 cells engineered via CRISPR/Cas9-mediated gene disruption to ablate expression of the ketohexokinase (KHK) gene. This knockout cell population serves as a loss-of-function model for investigating KHK-dependent biological processes, including fructose metabolism and its downstream effects on de novo lipogenesis. The polyclonal nature of this product provides a genetically diverse population, reducing clonal artifacts and enhancing the robustness of functional assays.
HAP1 is a near-haploid human cell line derived from the chronic myeloid leukemia (CML) line KBM-7. Originating from a male donor, HAP1 cells exhibit adherent growth and retain a single copy of most chromosomes, facilitating efficient gene targeting and knockout generation. The haploid genetic background of HAP1 simplifies functional genomics studies, making it an excellent host for investigating gene function in a hemizygous state without confounding wild-type alleles.
KHK catalyzes the phosphorylation of fructose to fructose-1-phosphate, the first step in fructose metabolism. This reaction bypasses the regulatory checkpoint of phosphofructokinase, leading to unconstrained flux into glycolysis and de novo lipogenesis. KHK expression is controlled by dietary fructose and hormonal signals such as insulin, acting through transcription factors ChREBP and SREBP-1c. The product fructose-1-phosphate is subsequently metabolized by aldolase B, generating dihydroxyacetone phosphate and glyceraldehyde. These intermediates can be converted by triokinase and glyceraldehyde kinase, ultimately fueling fatty acid synthesis via SREBP-1c and ChREBP. Thus, KHK sits at a critical juncture linking dietary fructose intake to lipid accumulation and metabolic disease. The knockout model disrupts this pathway, enabling detailed mechanistic studies.
In the context of HAP1 cells, which exhibit a leukemic metabolic profile, KHK knockout provides a unique platform to study fructose metabolism in cancer biology. The haploid nature ensures complete loss of function, avoiding wild-type compensation. This model is valuable for investigating KHK’s role in metabolic syndrome, NAFLD, and essential fructosuria, as well as for inhibitor screening. The absence of KHK activity allows assessment of fructose deprivation or supplementation on lipid accumulation and viability.
Researchers can use this polyclonal knockout population for western blotting to confirm KHK ablation, enzymatic activity assays for fructose-1-phosphate, and RT-qPCR for transcriptional analysis. Seahorse metabolic flux analysis quantifies glycolytic and oxidative shifts, while Oil Red O staining visualizes lipid droplets. These cells are suitable for high-throughput screening of fructose metabolism modulators and studying KHK’s role in cancer cell proliferation under fructose supplementation. For further information, contact Ascent Research.
