The PCCA Knockout Hep-G2 Cell Line is a CRISPR/Cas9-edited knockout cell line designed for targeted disruption of the PCCA gene in Hep-G2 hepatocellular carcinoma cells. This stable loss-of-function model enables investigation of propionyl-CoA carboxylase (PCC) alpha subunit function within a hepatic context. The engineered disruption abolishes catalytic conversion of propionyl-CoA to methylmalonyl-CoA, allowing study of metabolic pathway defects without overexpression artifacts. It is supplied as an adherent line, ready for standard culture protocols.
Hep-G2 is an adherent epithelial cell line derived from a well-differentiated hepatocellular carcinoma of a 15-year-old male, retaining wild-type p53 and numerous hepatic characteristics. This line serves as a foundational in vitro model for liver metabolism, toxicology, and oncogenesis research, exhibiting active expression of metabolic enzymes, transporters, and signaling networks relevant to hepatocyte physiology. The PCCA knockout in this background allows dissection of propionate catabolism within a human liver-derived system, facilitating comparison with primary hepatocytes and patient samples.
The PCCA gene encodes the alpha subunit of propionyl-CoA carboxylase, a mitochondrial biotin-dependent enzyme. It assembles with the PCCB beta subunit into the active heterododecamer. This complex catalyzes the carboxylation of propionyl-CoA to D-methylmalonyl-CoA, a pivotal step in degrading branched-chain amino acids and odd-chain fatty acids. Regulated by biotin availability and transcriptionally influenced by PPARA during lipid catabolism, PCCA-generated methylmalonyl-CoA is isomerized to succinyl-CoA by methylmalonyl-CoA mutase, feeding the TCA cycle. Knockout of PCCA disrupts this anaplerotic flux, causing accumulation of propionyl-CoA and odd-chain acyl intermediates, while depriving the TCA cycle of succinyl-CoA-derived carbons.
In the Hep-G2 background, PCCA knockout recapitulates propionic acidemia hallmarks, including impaired propionate metabolism and mitochondrial dysfunction. The hepatic context amplifies anaplerotic disruption, as TCA cycle function relies on amino acid?Cderived carbons. This model allows study of compensatory metabolic adaptations, such as glutaminolysis upregulation, and toxicity from propionate derivatives. The wild-type p53 status enables further evaluation of genotoxic stress under metabolite imbalance.
This knockout line is suited for modeling propionic acidemia-related metabolic acidosis, studying hepatic sensitivity to odd-chain fatty acid loads, and metabolic flux analysis using [13C]-propionate tracing. Researchers can combine LC-MS metabolomics, enzyme activity assays, Western blotting, and RT-qPCR to verify PCCA disruption and probe interconnected pathways like carbon metabolism. Cell viability assays under metabolic stress, such as high propionate or biotin depletion, aid therapeutic screening. For further technical details and custom solutions, please contact Ascent Research.
