The ALOX12 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from the human colorectal adenocarcinoma HT29 cell line. This model provides targeted disruption of the ALOX12 gene, encoding arachidonate 12-lipoxygenase, a key enzyme in eicosanoid synthesis. The polyclonal format retains genetic diversity while ensuring consistent loss of ALOX12 function, enabling robust studies of 12-lipoxygenase-dependent signaling in cancer biology. Researchers can employ these cells to dissect pathways regulated by 12-HETE production without clonal selection bias.
HT29 is a well-characterized intestinal epithelial cell line established from a primary colorectal adenocarcinoma of a 44-year-old female. With a hypertriploid karyotype, HT29 cells exhibit features of differentiated enterocytes, including polarized barrier formation and mucin production, and are widely used to model colorectal carcinogenesis, epithelial differentiation, and mucosal immunity. Harboring mutations in APC and p53, they recapitulate key oncogenic signaling abnormalities, making them a relevant platform for investigating tumor cell behavior and therapeutic interventions.
The ALOX12 gene product, arachidonate 12-lipoxygenase, oxygenates arachidonic acid to generate 12-HPETE, which is reduced to 12-HETE. 12-HETE acts via the GPR31 receptor to activate MAPK/ERK and PI3K/Akt cascades, modulating NF-??B-mediated inflammation, cell proliferation, and survival. Upstream regulators include STAT6, GATA1, and Wnt/??-catenin signaling, and its activity interacts with ALOX5, COX-2, and phospholipase A2. In HT29 cells, 12-HETE enhances integrin ??1-mediated functions; CRISPR/Cas9-mediated ALOX12 disruption abolishes this lipid mediator production, thereby silencing downstream signaling.
ALOX12 knockout in HT29 cells offers a valuable model to investigate the contribution of 12-HETE-GPR31 signaling to colorectal cancer progression. The absence of 12-HETE production in these APC-mutant, Wnt-active cells is expected to attenuate ERK and Akt phosphorylation, reduce proliferation, promote apoptosis, and alter inflammatory cytokine release. This system allows dissection of lipid mediator involvement in tumor cell autonomy, epithelial-mesenchymal transition, and the tumor microenvironment, highlighting vulnerabilities that could be exploited therapeutically.
Applications include functional characterization of ALOX12 in colorectal cancer, validation of the ALOX12/GPR31 axis as a drug target, and studies of 12-HETE-driven inflammatory and apoptotic mechanisms. Knockout confirmation can be achieved by Western blot, RT-qPCR, and LC-MS/MS for 12-HETE. Downstream signaling effects are assessed by phospho-ERK and phospho-Akt immunoblotting, while functional assays such as MTT viability, Annexin V apoptosis, Transwell migration, and colony formation gauge phenotypic changes. These polyclonal knockout cells are suited for high-throughput screening and mechanistic dissection. For technical inquiries, contact Ascent Research.
