The IDH3G Knouckout HT29 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal population derived from the HT29 human colorectal adenocarcinoma cell line, featuring targeted disruption of the IDH3G gene. This polyclonal knockout pool contains a heterogeneous mixture of edited alleles, avoiding clonal selection biases and enabling robust analysis of IDH3G loss-of-function in a cancer-relevant epithelial background. The knockout model is produced by CRISPR/Cas9-mediated gene disruption, resulting in a versatile tool for investigating IDH3G-dependent metabolic and signaling processes without the isolation of single-cell clones.
HT29 cells, originally isolated from a 44-year-old Caucasian female with colorectal cancer, are a well-established model for intestinal epithelial biology. These cells exhibit an undifferentiated phenotype under standard culture conditions but can be induced to undergo enterocytic differentiation, making them suitable for studying barrier function, drug transport, mucin secretion, and colorectal cancer pathophysiology. As a colorectal adenocarcinoma cell line, HT29 retains key oncogenic features and metabolic adaptations characteristic of tumor cells, providing a clinically relevant context for metabolic gene knockout studies.
IDH3G encodes the gamma regulatory subunit of mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH3), which catalyzes the oxidative decarboxylation of isocitrate to ??-ketoglutarate in the TCA cycle, coupled with the reduction of NAD+ to NADH. IDH3G functions as the regulatory arm of the heterotetrameric IDH3 complex, interacting with the catalytic IDH3A and IDH3B subunits to modulate enzymatic activity. Upstream, IDH3G expression is regulated by transcriptional coactivators PGC-1?? and NRF1 and is responsive to mTORC1 and HIF-1?? signaling. Downstream, IDH3G activity generates ??-ketoglutarate and NADH, fueling ATP synthesis via the electron transport chain and providing substrate for ??-ketoglutarate?Cdependent dioxygenases such as TET DNA demethylases and prolyl hydroxylases. Disruption of IDH3G therefore impairs TCA cycle flux, NADH regeneration, and cellular redox balance, with cascading effects on energy metabolism and epigenetic regulation.
In HT29 colorectal cancer cells, IDH3G knockout profoundly alters metabolic homeostasis. Given the reliance of proliferating cancer cells on TCA cycle intermediates and NADH for biosynthesis and energy production, loss of IDH3G is expected to reduce flux through the TCA cycle, diminish NAD+/NADH ratio, and perturb mitochondrial respiration. These metabolic disruptions can trigger compensatory mechanisms, affect cell proliferation, and sensitize cells to mitochondrial stress. The HT29 background also allows investigation of IDH3G in the context of intestinal differentiation and mucin production, providing insights into how mitochondrial metabolism interfaces with epithelial function in the colonic epithelium.
Researchers can employ this knockout pool for a broad spectrum of metabolic and cancer biology studies. Typical applications include TCA cycle flux analysis via LC-MS metabolomics, measurement of NAD+/NADH ratios, Seahorse metabolic flux assays, ATP and ROS quantification, and mitochondrial membrane potential assessment. The polyclonal nature is particularly suited for pooled functional screens, drug sensitivity profiling, and apoptosis or proliferation assays under metabolic stress. Western blotting and RT-qPCR enable confirmation of IDH3G disruption, while IDH3 enzymatic activity assays provide direct functional readouts. For further information or to customize this model for your specific research needs, please contact Ascent Research.
