The IVD knockout A-549 polyclonal cells are a CRISPR/Cas9-edited polyclonal population derived from the A-549 human lung adenocarcinoma cell line, engineered to disrupt the IVD gene encoding isovaleryl-CoA dehydrogenase. This product provides a loss-of-function model for studying leucine catabolism and organic acid metabolism in a human pulmonary epithelial context. The polyclonal format captures a mixture of edited alleles, enabling the study of heterogeneous knockout effects across the cell population. CRISPR/Cas9-mediated gene editing introduces targeted disruptions within the IVD locus, resulting in functional inactivation of the enzyme without reliance on single-cell clonal selection.
The A-549 host cell line is a widely utilized model of human lung adenocarcinoma, originally derived from the tumor tissue of a 58-year-old Caucasian male. These cells exhibit an epithelial-like morphology and a hypotriploid karyotype, and they retain features of alveolar type II pneumocytes, including surfactant production. A-549 cells are extensively employed in cancer biology, respiratory virus infection studies, and drug metabolism research, owing to their robust growth and well-characterized signaling networks. Their relevance to pulmonary physiology makes them particularly suitable for metabolic studies intersecting with lung disease or systemic metabolic disorders.
Isovaleryl-CoA dehydrogenase (IVD) is a mitochondrial flavoprotein that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA, a critical step in the leucine degradation pathway. This FAD-dependent reaction requires electron transfer flavoprotein (ETF) and ETF dehydrogenase for electron shuttling to the respiratory chain. IVD activity is transcriptionally regulated by factors such as PPARGC1A (PGC-1??), PPAR??, and HNF4A, which coordinate mitochondrial biogenesis and fatty acid oxidation. Upstream signals including leucine availability and glucagon modulate IVD expression, while its reaction product 3-methylcrotonyl-CoA is further metabolized by methylcrotonyl-CoA carboxylase (MCCC) to generate acetoacetate and acetyl-CoA, linking branched-chain amino acid catabolism to energy production and ketogenesis.
Loss of IVD function in A-549 cells recapitulates the metabolic block observed in isovaleric acidemia, an autosomal recessive organic aciduria characterized by accumulation of isovaleric acid and its conjugates. The A-549 background expression of lung epithelial markers enables investigation of tissue-specific consequences of IVD deficiency, including potential metabolic crosstalk with pulmonary surfactant synthesis and inflammatory responses. This knockout model provides a controlled system to examine the interplay between mitochondrial acyl-CoA dehydrogenase dysfunction and cellular stress pathways, without the confounding variables of patient-derived samples. Furthermore, the hypotriploid genome may influence the penetrance of heterozygous edits, making the polyclonal population a valuable tool for studying dose-dependent metabolic effects.
Researchers can employ these IVD knockout polyclonal cells to model isovaleric acidemia in vitro, perform enzymatic activity assays to confirm functional disruption, and quantify IVD mRNA and protein expression by RT-qPCR and Western blot. Targeted metabolomics via LC-MS allows detection of isovaleric acid and isovaleryl-CoA accumulation, providing a direct readout of pathway blockade. Functional studies, such as cell viability assays under leucine deprivation or Seahorse-based mitochondrial respiration analysis, reveal metabolic vulnerabilities and adaptive responses. Additionally, immunofluorescence staining for mitochondrial localization verifies enzyme mislocalization if targeting signals are affected. This knockout population supports drug screening for organic acidurias, biomarker discovery, and toxicity assays for branched-chain organic acids. For further technical specifications or to discuss custom applications, please contact Ascent Research.
