The BCKDK Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-mediated knockout population of HEK293T cells with targeted disruption of the BCKDK gene. BCKDK encodes branched-chain keto acid dehydrogenase kinase, a negative regulator of branched-chain amino acid (BCAA) catabolism. The polyclonal format provides a pooled loss-of-function model that circumvents clonal artifacts, enabling constitutive activation of the BCKDH complex for reproducible metabolic investigations.
The parental HEK293T cell line is a human embryonic kidney epithelial cell immortalized with sheared adenovirus 5 DNA and stably expressing the SV40 large T-antigen. This genetic background permits episomal plasmid replication and supports high-efficiency transient transfection, making it a standard host for recombinant protein production, lentivirus packaging, and CRISPR editing. Its kidney epithelial origin and robust growth characteristics offer a convenient model for studying epithelial metabolism.
BCKDK phosphorylates the E1?? subunit (BCKDHA) of the branched-chain ??-keto acid dehydrogenase (BCKDH) complex at Ser293 and Ser309, leading to complex inactivation and suppression of BCAA degradation. The kinase is allosterically inhibited by downstream ??-keto acid products (e.g., ??-ketoisocaproate) and regulated by the AMP/ATP ratio and hormones such as insulin and glucocorticoids. BCKDK physically interacts with BCKDH complex subunits (E1??, E1??, E2, E3) and the regulatory phosphatase PP2Cm, which counteracts its activity. By disrupting BCKDK, the BCKDH complex remains constitutively active, enhancing BCAA oxidation and altering signaling through pathways such as mTORC1 that sense leucine availability.
In the HEK293T model, BCKDK knockout provides an isogenic system to study the metabolic and signaling consequences of unchecked BCAA catabolism. This setup is valuable for investigating BCKDK deficiency-related neurodevelopmental disorders (autism, epilepsy, intellectual disability) and for exploring the role of BCAAs in insulin resistance and type 2 diabetes. The polyclonal nature ensures that results reflect consistent population-level responses, suitable for pharmacological screening.
Typical applications include spectrophotometric measurement of BCKDH activity, LC?CMS monitoring of BCAA and ??-keto acid levels, and western blotting for phospho-BCKDHA at Ser293/Ser309. Researchers can perform 13C-leucine metabolic flux analyses, insulin signaling assays (p-Akt Ser473, p-S6 Ser240/244), and cell viability or glucose uptake readouts. This knockout pool is also well-suited for testing small-molecule BCKDK inhibitors in preclinical metabolic disease research. For inquiries, please contact Ascent Research.
