The ACO1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population derived from HAP1 cells, with targeted disruption of the ACO1 gene. This model enables loss-of-function studies of the bifunctional ACO1 protein, which operates as cytoplasmic aconitase in the tricarboxylic acid (TCA) cycle and as iron regulatory protein 1 (IRP1) controlling cellular iron homeostasis. The polyclonal composition, arising from heterogeneous editing events, provides a genetically defined system for investigating ACO1 function without the necessity of single-cell cloning.
HAP1 is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia model. Its mostly haploid genome simplifies loss-of-function studies, eliminating heterozygous confounding effects. Retaining BCR-ABL dependency, HAP1 provides a relevant hematopoietic context for investigating leukemia biology and hematopoiesis pathways, making it an ideal host for ACO1 knockout studies.
ACO1 encodes a dual-function protein that interconverts between a [4Fe-4S] cluster-containing aconitase and an apo-IRP1 with high affinity for iron-responsive elements (IREs). Under iron-replete conditions, the cluster, assembled by ISCU/NFS1/frataxin, enables TCA cycle catalysis. Iron deficiency, oxidative stress, or nitric oxide causes cluster loss, activating IRP1. IRP1 binds IREs in mRNAs, stabilizing transferrin receptor (TFRC) transcript and blocking ferritin (FTL/FTH1) translation, while also upregulating the importer SLC11A2 and repressing the exporter SLC40A1. Thus, ACO1 coordinates iron uptake, storage, and export, and its disruption profoundly impacts cellular iron distribution and aconitase activity.
In HAP1 leukemia cells, loss of ACO1 disrupts iron homeostasis and metabolic activity, making this model highly relevant for uncovering iron dependencies in cancer. Perturbed IRP1 signaling is expected to alter TFRC surface presentation, ferritin protein levels, and cellular iron flux, while diminished aconitase activity may impair mitochondrial respiration. These changes can modulate sensitivity to ferroptosis, an iron-catalyzed cell death pathway, and influence leukemogenesis, positioning the knockout as a powerful tool for studying iron addiction and redox vulnerability in hematopoietic malignancies.
Key applications include Western blot detection of IRP1 and downstream targets such as TFRC and ferritin, RT-qPCR for iron-responsive gene expression, iron uptake assays, and aconitase activity measurements. Electrophoretic mobility shift assays (EMSA) validate IRE-binding loss, while flow cytometry monitors surface TFRC levels. Cell viability and lipid peroxidation assays under ferroptosis-inducing conditions (e.g., erastin) reveal ACO1-dependent sensitivities. This polyclonal knockout model supports drug screening, CRISPR validation, and iron metabolism research. For more information, contact Ascent Research.
