IDH3A Knockout HEK293T Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout cell population for loss-of-function investigation of the IDH3A gene. This product comprises a heterogeneous pool of HEK293T cells with targeted disruption of the IDH3A locus, enabling researchers to assess gene function without the confounding effects of clonal selection. The polyclonal nature captures a broad range of editing events, offering a robust model for studying mitochondrial metabolism and TCA cycle dynamics in a physiologically relevant epithelial context.
The HEK293T host cell line is a derivative of the human embryonic kidney HEK293 line, stably expressing the SV40 large T antigen. This characteristic renders the cells highly transfectable and supports high-level protein expression and viral production, making HEK293T a universal workhorse in molecular biology and biotechnology. Originating from fetal kidney epithelium, these adherent cells retain active metabolic pathways and are extensively used in studies requiring robust mitochondrial function and rapid proliferation.
IDH3A encodes the alpha catalytic subunit of the mitochondrial NAD-dependent isocitrate dehydrogenase (IDH3), a critical allosteric enzyme complex that oxidatively decarboxylates isocitrate to alpha-ketoglutarate (??-KG) with concurrent reduction of NAD+ to NADH. This reaction is a rate-limiting step of the tricarboxylic acid (TCA) cycle and directly couples carbon metabolism to electron transport chain activity. IDH3A functions within a heterotetrameric complex comprising IDH3B and IDH3G subunits and interacts with multiple mitochondrial matrix proteins and TCA cycle enzymes, including citrate synthase and aconitase. Its activity is governed by upstream regulators such as AMPK, PGC-1??, NRF1, NRF2, and substrate availability (isocitrate, NAD+), while the generated ??-KG serves as a co-substrate for ??-KG-dependent dioxygenases, including prolyl hydroxylases that regulate HIF stability, and histone and DNA demethylases, thereby linking TCA cycle flux to epigenetic remodeling and cellular oxygen sensing.
Knockout of IDH3A in HEK293T cells disrupts the primary oxidative decarboxylation step of the TCA cycle, leading to accumulation of isocitrate and depletion of ??-KG and NADH. As HEK293T cells rely heavily on oxidative metabolism, this perturbation attenuates mitochondrial respiration and ATP synthesis, mimicking mitochondrial dysfunction phenotypes. The polyclonal knockout pool further models heterogeneous loss-of-function effects, making it suitable for population-level analyses of metabolic adaptation, metabolic reprogramming, and compensatory pathways. This system is therefore valuable for exploring the multifaceted roles of IDH3A in energy homeostasis, redox balance, and metabolite-sensitive signaling cascades within a well-characterized epithelial background.
This product is ideally suited for a wide range of experimental applications, including validation by Western blotting or RT-qPCR, functional assessment of mitochondrial respiration using Seahorse metabolic flux assays, quantification of ??-KG and NADH levels, cell viability assays under metabolic stress, and transcriptomic profiling via RNA-seq. It serves as a powerful tool for investigating TCA cycle dysfunction, metabolic reprogramming in cancer, mitochondrial disease mechanisms, and the impact of ??-KG on epigenetic landscapes. For more details or to inquire about this product, please contact Ascent Research.
