The IDH2 Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population targeting the IDH2 gene in HT29 colorectal adenocarcinoma cells. Generated by transient delivery of Cas9 and guide RNA, the polyclonal pool contains diverse loss-of-function alleles without single-cell isolation, reducing clonal bias and enabling functional assessment of IDH2 disruption across a heterogeneous cancer cell background.
The HT29 human colorectal adenocarcinoma cell line serves as a widely used intestinal epithelial model. Originating from a primary colon tumor, HT29 cells display epithelial characteristics such as polarization, mucus production, and expression of drug transporters, making them particularly suitable for studying colorectal cancer metabolism, intestinal drug transport, and chemotherapeutic sensitivity.
IDH2 encodes mitochondrial isocitrate dehydrogenase that converts isocitrate to ??-ketoglutarate (??-KG) and NADP+ to NADPH, serving as a critical source of mitochondrial NADPH for redox homeostasis. IDH2 is regulated upstream by SIRT3, PGC-1??, and AMPK, and functions as a homodimer within the TCA cycle. Its product ??-KG is a co-substrate for dioxygenases that control HIF stability (prolyl hydroxylases) and epigenetic marks (TET DNA demethylases, JmjC histone demethylases). Mutant IDH2-derived 2-hydroxyglutarate inhibits these enzymes, highlighting the role of wild-type IDH2 in maintaining normal metabolism and gene regulation.
In HT29 colorectal cancer cells, IDH2 knockout disrupts TCA cycle flux, impairs ??-KG and NADPH production, and perturbs cellular redox balance. The loss of IDH2-driven NADPH generation compromises antioxidant defenses and alters bioenergetics, while reduced ??-KG availability diminishes dioxygenase activity, potentially stabilizing HIF-?? and causing aberrant DNA and histone methylation patterns. These molecular changes enable the study of metabolic rewiring, oxidative stress responses, and epigenetic dysregulation in a colon cancer context.
Typical research uses include dissecting mitochondrial metabolism in colorectal adenocarcinoma, probing IDH2-dependent NADPH production and antioxidant mechanisms, investigating ??-KG-dependent dioxygenase functions (HIF prolyl hydroxylases, TET, JmjC families), assessing drug metabolism and resistance, and exploring functional compensation between cytoplasmic IDH1 and mitochondrial IDH2. The cells are compatible with Western blotting, RT-qPCR, RNA-seq, metabolomic analyses (??-KG, 2-HG, NADPH/NADP+), Seahorse respirometry, ROS measurements, cell proliferation and apoptosis assays, and ChIP-seq for histone modifications. For inquiries, contact Ascent Research.
