The IVD Knockout HAP1 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population targeting IVD (isovaleryl-CoA dehydrogenase) in HAP1 near-haploid human cells. Generated via CRISPR/Cas9-mediated gene disruption, this loss-of-function model eliminates functional IVD without single-clone selection, maintaining genetic diversity while ensuring robust locus targeting for leucine catabolism and mitochondrial metabolism studies.
HAP1 cells are a near-haploid human line derived from KBM-7 chronic myeloid leukemia cells, exhibiting fibroblast-like morphology and p53 deficiency. The near-haploid karyotype simplifies genetic manipulation, as only one allele needs disruption for phenotypic expression, facilitating knockout model generation. Combined with robust growth, HAP1 is ideal for functional genomics and metabolic studies, unmasking recessive phenotypes linked to enzyme deficiencies.
IVD encodes a mitochondrial matrix flavoenzyme that catalyzes the dehydrogenation of isovaleryl-CoA to 3-methylcrotonyl-CoA, a key step in leucine degradation, and transfers electrons to electron transfer flavoprotein (ETF) for subsequent ATP production via the respiratory chain. Its expression is transcriptionally regulated by PPARA and PPARGC1A downstream of AMPK signaling, responding to leucine substrate availability. The reaction product 3-methylcrotonyl-CoA is further metabolized by methylcrotonyl-CoA carboxylase (MCC) into acetyl-CoA and ketone bodies through HMG-CoA lyase. Disruption of IVD results in accumulation of isovaleryl-CoA and its toxic derivative isovaleric acid, recapitulating the metabolic hallmark of isovaleric acidemia.
In HAP1 cells, IVD knockout provides a robust model of isovaleric acidemia, an autosomal recessive organic aciduria. The near-haploid background eliminates allelic compensation, enhancing phenotype penetrance, while p53 deficiency may influence metabolic stress responses. This model enables detailed studies of mitochondrial dysfunction, leucine-dependent toxicity, and metabolic reprogramming in branched-chain amino acid disorders, and is ideal for screening compounds that alleviate toxic intermediate buildup.
This polyclonal knockout population supports diverse assays for leucine metabolism and therapeutic investigation. Western blotting and RT-qPCR confirm IVD loss, while enzyme activity assays and LC-MS metabolomics validate isovaleryl-CoA accumulation. Seahorse mitochondrial stress tests assess oxidative respiration changes, and leucine-challenge viability assays quantify substrate sensitivity. Applications include metabolic engineering, drug discovery for organic acidurias, and mitochondrial biology. For further information, contact Ascent Research.
