The HADHB Knockout HT29 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population with targeted disruption of the HADHB gene, designed for loss-of-function analyses. This heterogeneous pool of HT29 cells lacks expression of the mitochondrial trifunctional protein ??-subunit, enabling robust gene knockout studies without clonal selection biases. The polyclonal format is optimal for population-level assays investigating the gene??s role in fatty acid metabolism and colorectal cancer biology.
The HT29 host cell line originates from a human colorectal adenocarcinoma of a 44-year-old female and serves as a widely used in vitro model of intestinal epithelial cells. These cells are instrumental for studying colorectal carcinoma pathogenesis, epithelial barrier function, and cellular metabolism, retaining features such as polarization and differentiation. Their robust growth and defined genetic background provide a reliable platform for generating gene knockouts to dissect colorectal cancer biology.
HADHB encodes the ??-subunit of mitochondrial trifunctional protein (MTP), catalyzing long-chain fatty acid ??-oxidation. In a complex with HADHA, it converts long-chain acyl-CoA into acetyl-CoA, NADH, and short-chain acyl-CoA, driving ATP production. Transcription is regulated by PPAR??, PPAR??, and PGC-1??, activated by AMPK and SIRT1. Within ??-oxidation, HADHB functions downstream of CPT1A, CPT2, and VLCAD, and upstream of HADH, serving as a critical node in lipid catabolism and energy homeostasis.
In HT29 cells, HADHB knockout disrupts mitochondrial fatty acid oxidation, impairing acetyl-CoA and NADH generation, reducing ATP levels, and altering lipid intermediates. This metabolic reprogramming affects proliferation, survival, and stress responses. Given the reliance of cancer cells on lipid metabolism for membrane synthesis and redox balance, this model is valuable for dissecting mitochondrial ??-oxidation contributions to tumorigenesis, drug resistance, and metabolic plasticity in colorectal carcinoma.
The model supports assays including western blotting and RT-qPCR for knockout confirmation, labeled palmitate oxidation assays, and Seahorse mitochondrial stress tests. ATP luminescence quantifies energy deficits, while proliferation, migration, and invasion assays reveal phenotypic effects. Oil Red O staining visualizes lipid accumulation. Applications include studying lipid metabolism in colorectal cancer, mitochondrial dysfunction, and therapeutic target identification. For further information, contact Ascent Research.
