The ATAD3A Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-mediated polyclonal knockout population in which the ATAD3A gene has been disrupted in HT29 human colorectal adenocarcinoma cells. This polyclonal pool offers a loss-of-function model that mitigates clonal artifacts and captures the range of knockout phenotypes, suitable for pooled screening and population-based assays.
The HT29 cell line originates from a colorectal adenocarcinoma of a 44-year-old female and is widely used for studying colon cancer, epithelial differentiation, and intestinal barrier function. HT29 cells can undergo enterocytic differentiation, making them a valuable model for examining oncogenic signaling and therapeutic responses in a physiologically relevant epithelial context.
ATAD3A is an inner mitochondrial membrane ATPase that critically regulates mitochondrial dynamics and ER?Cmitochondria contact sites. It interacts with mitochondrial fusion proteins MFN1 and MFN2 and the fission factor DRP1, and forms complexes with VAPB and SLC25A1 at ER?Cmitochondria interfaces to facilitate cholesterol transport by binding StAR and activating CYP11A1. ATAD3A expression is driven by transcription factors c-Myc, SP1, and PGC-1??, and is induced by oxidative and ER stress. Loss of ATAD3A disrupts DRP1-mediated fission and MFN1/2-mediated fusion, causing aberrant mitochondrial morphology, impaired cholesterol trafficking, and altered ??-catenin signaling.
In HT29 cells, ATAD3A knockout is instrumental for dissecting tumor metabolic vulnerabilities linked to cholesterol metabolism and mitochondrial adaptation. Disruption of ATAD3A??s interactions with DRP1 and MFN2 perturbs mitochondrial fission-fusion balance, processes often dysregulated in colorectal cancer. Furthermore, impaired ER?Cmitochondria cholesterol transport via CYP11A1 and StAR underscores the model??s relevance to lipid metabolic reprogramming. This system allows investigation of how ATAD3A deficiency impacts proliferation, differentiation, and apoptosis in colorectal adenocarcinoma.
Experimental applications include immunofluorescence for mitochondrial morphology, Western blot of DRP1, MFN1, and MFN2, cholesterol efflux assays, and CYP11A1 quantification. Proliferation (MTT) and apoptosis (Annexin V) assays, RT-qPCR for target genes like ??-catenin, and Seahorse mitochondrial stress tests enable functional and metabolic profiling. These studies help elucidate ATAD3A??s roles in colorectal cancer signaling, drug resistance, and metabolic reprogramming. For additional information, please contact Ascent Research.
