The ASAH1 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population generated in the HT-29 human colorectal adenocarcinoma line, featuring targeted disruption of the ASAH1 gene. This loss-of-function model eliminates acid ceramidase activity, preventing the lysosomal hydrolysis of ceramide into sphingosine and free fatty acid. The polyclonal nature ensures a range of genetic modifications, providing a robust platform for studying ASAH1 deficiency without clonal selection bias.
The HT-29 host cell line was originally isolated from a primary colon adenocarcinoma of a 44-year-old Caucasian female. HT-29 cells exhibit an adherent, epithelial morphology and are capable of forming polarized monolayers with tight junctions, mimicking key aspects of the intestinal epithelium. When appropriately differentiated, they secrete mucin and express enterocytic markers, serving as a model for barrier function, drug absorption, and nutrient transport.
ASAH1 encodes acid ceramidase, a lysosomal enzyme that catalyzes the deacylation of ceramide to generate sphingosine, the immediate precursor of the pro-survival signaling lipid sphingosine-1-phosphate (S1P). Acid ceramidase activity is allosterically activated by the saposin D cofactor and is regulated by lysosomal pH; its expression is transcriptionally upregulated by pro-inflammatory cytokines such as TNF-??. Ceramide itself is synthesized by ceramide synthases (CerS1?C6) or released from sphingomyelin by sphingomyelinases, functioning as a pro-apoptotic messenger. Sphingosine is phosphorylated by sphingosine kinases SPHK1 and SPHK2 to produce S1P, which signals through five G protein-coupled S1P receptors (S1PR1?C5) to promote cell survival and migration. Consequently, ASAH1 sits at a critical junction that balances ceramide-induced apoptosis with S1P-mediated pro-survival pathways.
In the context of HT-29 colorectal adenocarcinoma cells, disruption of ASAH1 is anticipated to elevate intracellular pro-apoptotic ceramide levels while reducing S1P production, thereby sensitizing the cells to apoptotic stimuli. This altered lipid profile provides a cellular system to dissect ceramide-dependent cell death mechanisms and their intersection with oncogenic signaling in colorectal cancer. Additionally, because HT-29 cells retain properties of intestinal epithelial cells, the knockout model enables studies of how ASAH1 deficiency influences mucosal barrier integrity, inflammatory responses, and the sphingolipid rheostat in gut physiology. It may also serve as a model for Farber disease, enabling investigation of ASAH1 deficiency in an epithelial background.
This polyclonal knockout cell product is suitable for a broad range of downstream applications. Researchers can quantify ceramide accumulation by LC-MS, measure sphingosine and S1P levels via ELISA, and assess apoptosis using Annexin V/PI staining. Cell viability and migration assays can evaluate chemosensitization in colorectal cancer contexts, while RNA-seq analysis enables global transcriptomic profiling of sphingolipid pathway perturbations. The model also supports studies of intestinal barrier function, inflammatory bowel disease, and sphingolipid-related neurodegeneration. For more information about customizing this product or discussing further experimental possibilities, please contact Ascent Research.
