The BCKDK Knockout HeLa Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population of HeLa cells with targeted disruption of the BCKDK gene, creating a loss-of-function model. This mixed knockout pool avoids clonal biases and is suited for pooled screening and functional assays that benefit from heterogeneous genetic backgrounds.
HeLa cells, an HPV18-positive cervical adenocarcinoma line derived from a 31-year-old female, express E6 and E7 oncoproteins that inactivate p53 and RB, ensuring robust and stable proliferation. Widely employed in cancer biology and virology, their well-characterized genome and transfection efficiency make them an ideal host for metabolic pathway dissection.
BCKDK is a mitochondrial kinase that phosphorylates BCKDHA at Ser293, inactivating the BCKDH complex and reducing branched-chain keto acid oxidation. This leads to accumulation of BCAAs (leucine, isoleucine, valine) and their keto acids, which activate mTORC1 signaling via S6K1 and 4E-BP1. Upstream, BCKDK is regulated by AMPK and insulin/PI3K/AKT pathways, while PPM1K phosphatase reverses the phosphorylation. The BCKDH complex includes BCKDHB, DBT, and DLD, and its modulation balances nutrient signals with cell growth and autophagy.
In the HeLa context, where mTORC1 is constitutively active, BCKDK knockout is expected to de-repress BCKDH, lowering BCAA levels and attenuating mTORC1 activity. This may expose metabolic vulnerabilities, particularly under nutrient limitation, and clarify how BCAA-driven anabolic signaling supports the transformed phenotype.
These polyclonal knockout cells enable investigation of BCAA metabolism and mTORC1 regulation via phospho-BCKDHA (Ser293) western blotting, BCKDH activity assays, and LC-MS quantification of BCAA/BCKA levels. Functional readouts include phospho-S6K1/4E-BP1 analysis, cell proliferation under BCAA deprivation, Seahorse metabolic flux, and apoptosis assays. The model supports drug target characterization and metabolic reprogramming studies. For further technical support, contact Ascent Research.
