The BCKDK Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the near-haploid human HAP1 cell line. This product introduces targeted gene disruption of BCKDK, which encodes the branched-chain ketoacid dehydrogenase kinase, a critical regulator of branched-chain amino acid (BCAA) catabolism. The polyclonal format captures a diverse pool of gene-edited cells, enabling robust population-level functional studies without clonal selection.
HAP1 cells originate from the KBM-7 chronic myeloid leukemia (CML) line and maintain a near-haploid karyotype, making them a leading model for functional genomics and CRISPR-based screens. Their adherent, fibroblast-like morphology and stable haploid state minimize confounding effects from homologous chromosomes, facilitating efficient knockout generation and phenotypic analysis. This background is widely employed in metabolic and signaling research.
BCKDK functions as a negative regulator of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex by phosphorylating serine residues on the E1?? subunit, leading to enzyme inactivation and reduced BCAA oxidative decarboxylation. This kinase is activated by elevated BCAA levels, insulin, and nutrient status, while its activity is reversed by the phosphatase PPM1K. BCKDK directly interacts with the BCKDH complex subunits E1??, E1??, E2, and E3, positioning it as a central node in BCAA metabolism. Downstream targets include BCKDH activity, cellular BCAA concentrations, and mTORC1 signaling. Key pathway components encompass BCKDK, BCKDH, BCAAs (leucine, isoleucine, valine), branched-chain alpha-keto acids, and PPM1K.
Disruption of BCKDK in HAP1 cells removes the inhibitory constraint on BCKDH, leading to constitutive BCAA catabolism. This haploid knockout model recapitulates molecular features of maple syrup urine disease (MSUD) and branched-chain ketoacid dehydrogenase kinase deficiency, offering a precise tool for dissecting disease mechanisms. The polyclonal population allows assessment of phenotypic heterogeneity and is particularly suited for pooled CRISPR modifier screens and metabolic perturbation studies.
This product supports diverse research applications, including metabolic disease modeling, mTOR signaling analysis in response to amino acid availability, kinase inhibitor profiling, and optimization of CRISPR screening workflows in HAP1 cells. Representative assays include western blotting for BCKDK and phosphorylated BCKDH, quantitative BCAA measurement, BCKDH enzyme activity assays, metabolic flux analysis, and cell viability under BCAA-restricted conditions. For further technical information, please contact Ascent Research.
