HEXB Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from the human HT29 colorectal adenocarcinoma cell line. Targeted disruption of the HEXB gene via CRISPR/Cas9 produces a heterogeneous pool of cells lacking functional HEXB protein, thereby eliminating both ??-hexosaminidase A (HEXA/HEXB heterodimer) and B (HEXB homodimer) enzymatic activities. This model facilitates investigation of HEXB deficiency within an epithelial cancer background.
The HT29 cell line is a well-characterized colorectal adenocarcinoma model exhibiting epithelial morphology, mucin production, and adherent growth. Widely employed in colon cancer research and intestinal differentiation studies, HT29 cells retain features of intestinal epithelium and are amenable to diverse experimental manipulations. Their colorectal origin renders them particularly suitable for exploring links between lysosomal function, glycoconjugate metabolism, and tumor biology. The HEXB knockout in this context enables studies of how disrupted ganglioside degradation influences cancer cell physiology.
HEXB encodes the beta subunit of lysosomal ??-hexosaminidase, which forms heterodimers with HEXA to generate ??-hexosaminidase A or homodimers for ??-hexosaminidase B. These enzymes cleave terminal N-acetylhexosamine residues from GM2 gangliosides, GA2 globoside, and other glycoconjugates. Upstream, transcription factors TFEB, MITF, and TFE3 regulate HEXB expression in response to nutrient-sensing signals, coordinating lysosomal biogenesis. Downstream, HEXB deficiency blocks substrate degradation, leading to accumulation that phenocopies the biochemical hallmark of Sandhoff disease. Efficient substrate processing requires interaction with HEXA and the GM2 activator protein GM2A.
In HT29 colorectal adenocarcinoma cells, HEXB knockout provides a unique model to interrogate the intersection of lysosomal storage pathology and cancer. HT29 cells possess active endolysosomal pathways; loss of HEXB induces abnormal lysosomal accumulation of gangliosides, which can alter membrane composition, signaling cascades, and cellular homeostasis. This allows investigation of how ganglioside storage impacts colorectal cancer cell behavior, including epithelial barrier integrity, differentiation, and chemotherapeutic response. Moreover, the model enables examination of TFEB/MITF-driven lysosomal adaptation in tumor cells under storage stress.
The HEXB Knockout HT29 Polyclonal Cells support diverse applications, including mechanistic studies of Sandhoff disease, high-throughput screening for enzyme replacement therapies or pharmacological chaperones, and evaluation of substrate reduction strategies. Representative assays encompass fluorometric ??-hexosaminidase activity assays, LC-MS-based quantification of GM2 ganglioside, HEXB western blotting, LysoTracker staining for lysosomal mass, and LAMP1 immunofluorescence. Cell viability readouts under substrate accumulation conditions can assess therapeutic interventions. This polyclonal knockout population serves as a robust model for both lysosomal storage disorder research and colorectal cancer lysosomal biology. For further information, please contact Ascent Research.
