The ACO1 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population of human embryonic kidney HEK293T cells, engineered for disruption of the ACO1 gene. This product provides a heterogeneous pool of cells carrying diverse loss-of-function mutations, eliminating the need for single-cell cloning while enabling robust assessment of IRP1/cytosolic aconitase function in bulk populations. As a polyclonal knockout reagent, it supports biochemical, transcriptomic, and functional assays where population-level effects are of interest.
HEK293T cells are an epithelial adherent line derived from HEK293 cells, with stable integration of the SV40 large T antigen. This modification enhances episomal replication of plasmids containing the SV40 origin, making HEK293T a preferred host for recombinant protein overexpression, lentiviral packaging, and high-titer viral production. Their human origin, rapid growth, and high transfectability render them particularly suitable for genetic perturbation studies, including functional genomics screens and pathway dissection under physiologically relevant conditions.
The ACO1 gene encodes a bifunctional protein that, under high intracellular iron, assembles a [4Fe-4S] cluster to function as cytosolic aconitase, catalyzing the interconversion of citrate and isocitrate. In iron-deficient conditions, loss of the iron-sulfur cluster converts the protein into iron regulatory protein 1 (IRP1), which binds iron-responsive elements (IREs) in the untranslated regions of mRNAs encoding key iron metabolism proteins. This post-transcriptional regulation modulates the expression of transferrin receptor (TFRC), ferritin subunits (FTL and FTH1), ferroportin (SLC40A1), DMT1 (SLC11A2), and other targets. ACO1 is activated by cellular iron depletion, hypoxia via HIF1A, nitric oxide, and reactive oxygen species, and it interacts with IRP2 (IREB2), the E3 ubiquitin ligase FBXL5, and the cytosolic iron-sulfur assembly (CIA) complex. Disruption of ACO1 therefore abolishes both aconitase activity and IRE-mediated control, leading to dysregulated iron uptake, storage, and export.
In the HEK293T background, ACO1 knockout provides a clean platform to interrogate IRP-dependent and aconitase-dependent processes without interference from endogenous IRP1. The robust protein expression and viral packaging capacity of these cells allows for complementation studies, overexpression of mutant IRP1 variants, or introduction of iron-responsive reporters. This model is particularly valuable for dissecting the interplay between cellular iron status, citrate metabolism, and responses to oxidative stress, as well as for exploring ferroptosis sensitivity, given the role of iron in lipid peroxidation. The polyclonal nature ensures that off-target editing does not confound population-level conclusions in appropriately controlled experiments.
Researchers can employ these knockout cells in a wide array of applications, including Western blot analysis of IRP1 and downstream targets (TFRC, ferritin), RT-qPCR of IRE-containing transcripts, aconitase enzymatic activity assays, radioactive 55Fe uptake measurements, RNA immunoprecipitation for IRE binding, and intracellular iron quantification by ferrozine assay. The model aids investigations into iron-refractory iron deficiency anemia, neurodegenerative disorders such as Friedreich ataxia, aceruloplasminemia, and anemia of chronic disease. Additionally, it serves as a tool for studying hypoxia signaling (HIF2A/EPAS1 regulation) and the crosstalk between iron homeostasis and ferroptosis. For technical inquiries or custom applications, please contact Ascent Research.
