ACADM Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HAP1 cell line, engineered to disrupt the human ACADM gene. This product eliminates medium-chain acyl-CoA dehydrogenase (MCAD) function, providing a pooled loss-of-function model for studying mitochondrial fatty acid ??-oxidation without the biases of single-cell cloning. The polyclonal format maintains genetic diversity while ensuring robust target-gene inactivation, making it well-suited for population-level phenotypic analyses and high-content metabolic screens.
The HAP1 cell line is a near-haploid, male-derived, semi-adherent cell line originally isolated from the KBM-7 chronic myeloid leukemia (CML) line. Owing to its haploid karyotype in over 90% of cells, HAP1 is a widely adopted host for CRISPR-based genetic engineering, as single-copy gene disruption yields straightforward loss-of-function phenotypes without requiring zygosity selection. Despite its transformed origin, HAP1 retains essential signaling pathways and metabolic machinery, making it a tractable system for functional genomics studies in a human background.
ACADM encodes medium-chain acyl-CoA dehydrogenase (MCAD), which catalyzes the first step of mitochondrial ??-oxidation of medium-chain (C4?CC12) fatty acids, converting acyl-CoAs to enoyl-CoAs and transferring electrons to electron transfer flavoprotein (ETF). MCAD is transcriptionally activated by PPAR??/PGC-1?? and is modulated by SIRT1 and AMPK in response to fasting. The enzyme’s activity generates acetyl-CoA, NADH, and FADH2, which fuel the TCA cycle and oxidative phosphorylation, and contributes to ketone body formation during catabolic states. Key interacting partners include ETF, ETF dehydrogenase (ETFDH), and the mitochondrial trifunctional protein, while pathway components such as CPT1A and SLC25A20 facilitate substrate delivery to the ??-oxidation spiral.
In HAP1 cells, ACADM knockout impairs metabolism of medium-chain fatty acids, resulting in accumulation of medium-chain acylcarnitines (octanoylcarnitine and hexanoylcarnitine) and diminished ketogenesis and ATP synthesis from lipid substrates. Under fatty acid load or glucose deprivation, these cells experience energetic stress and lipotoxicity, mirroring key metabolic derangements seen in medium-chain acyl-CoA dehydrogenase deficiency (MCADD). This model thus offers a human cellular system to investigate mitochondrial fatty acid oxidation defects and their downstream consequences.
This knockout model is suited for a broad range of metabolic research applications, including dissection of mitochondrial ??-oxidation pathways, MCADD disease modeling, and evaluation of pharmacochaperones or read-through compounds. Researchers can employ acylcarnitine profiling by LC-MS/MS, flux assays using [3H]palmitate or BODIPY-C12, and ketone body (??-hydroxybutyrate) measurements to quantify lipid handling defects. In addition, ATP luminescence assays and Seahorse respirometry reveal bioenergetic impairment, while viability assays under nutrient stress enable high-throughput screening of metabolic modulators. For further details, please contact Ascent Research.
