The BCKDK Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population designed to disrupt BCKDK expression in the HT29 human colorectal adenocarcinoma line. This loss-of-function model eliminates inhibitory phosphorylation of the BCKDH complex, enabling direct investigation of BCKDK’s role in branched-chain amino acid (BCAA) catabolism and mTORC1 regulation. The polyclonal format ensures diverse allelic disruptions, providing a robust system for studying metabolic reprogramming without clonal artifacts.
HT29 cells, derived from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female, are epithelial cells capable of enterocytic differentiation. Widely used in cancer and intestinal biology research, they provide a relevant background for studying BCAA metabolism in colorectal cancer. Their well-characterized signaling networks and differentiation potential allow exploration of BCKDK function in both proliferative and differentiated states.
BCKDK is a kinase that inactivates the BCKDH complex by phosphorylating its E1?? subunit at Ser293/Ser303, suppressing BCAA catabolism. This phosphorylation is regulated by branched-chain ??-keto acids, PPAR?? agonists, and insulin. BCKDK interacts with BCKDH E2, and its action elevates BCAAs (leucine, isoleucine, valine), which activate mTORC1. Consequently, BCKDK serves as a metabolic switch connecting BCAA degradation to nutrient signaling; its loss leads to constitutive BCKDH activity, BCAA depletion, and mTORC1 inhibition.
In the HT29 context, BCKDK knockout forces continuous BCAA degradation, likely lowering mTORC1 activity and impacting cell proliferation and differentiation. This metabolic shift provides a model to examine how BCAA metabolic control influences colorectal cancer growth and epithelial differentiation. Additionally, the knockout cells enable dissection of mTORC1-dependent vs. -independent effects of BCKDK, offering insights into metabolic vulnerabilities in tumors.
These polyclonal knockout cells are suited for BCAA quantification via LC?MS, BCKDH activity assays, mTORC1 signaling analysis (phospho?S6K1), and cell proliferation/differentiation assays. They can be used in drug screens targeting BCKDK or BCAA metabolism and in transcriptomic studies (RNA?seq). For further information or technical assistance, please contact Ascent Research.
