The BCKDHB Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population carrying targeted gene disruption of BCKDHB in human embryonic kidney HEK293T cells. This loss-of-function model is designed for investigating branched-chain amino acid (BCAA) catabolism and its role in metabolic disorders.
The HEK293T cell line, derived from HEK293 cells and stably expressing the SV40 large T antigen, is widely used for protein expression and viral production due to its high transfection efficiency and robust growth. These characteristics, combined with its epithelial origin and intact mitochondrial metabolism, make it an ideal platform for dissecting metabolic pathways.
BCKDHB encodes the E1?? subunit of the mitochondrial branched-chain ??-keto acid dehydrogenase (BCKDH) complex, which catalyzes the oxidative decarboxylation of branched-chain ??-keto acids (KIC, KMV, KIV) derived from leucine, isoleucine, and valine. This rate-limiting reaction channels carbon into the TCA cycle via acetyl-CoA and succinyl-CoA, producing NADH and ATP, and supplies precursors for neurotransmitter biosynthesis. BCKDH activity is regulated by upstream signals such as KLF15, PPARGC1A, insulin, and glucagon, and the complex assembly requires interactions with BCKDHA, DBT, and DLD. Reversible phosphorylation by BCKDK and PPM1K fine-tunes enzymatic activity. Disruption of BCKDHB abolishes complex function, causing BCKA accumulation and metabolic disruption analogous to maple syrup urine disease type 1B.
In HEK293T cells, which retain active BCAA oxidation pathways, BCKDHB knockout recapitulates the metabolic blockade seen in branched-chain ketoaciduria. This model facilitates study of mitochondrial stress responses, nutrient signaling crosstalk, and the impact of BCAA metabolic defects on cellular energetics and neurotoxicity.
Key applications include quantifying BCAA and BCKA levels by LC-MS/MS, measuring BCKDH enzymatic activity spectrophotometrically, assessing mitochondrial respiration with Seahorse analyzers, and performing RT-qPCR for BCAA metabolic genes. Additionally, leucine stress viability assays and Western blotting for BCKDHB enable functional validation. These tools support drug screening for metabolic encephalopathies and mechanistic dissection of BCAA-related mitochondrial biology. For further details, please contact Ascent Research.
