The DLGAP5 Knockout HT29 Polyclonal Cells product is a genetically modified cell population derived from the HT29 colorectal adenocarcinoma cell line, engineered using CRISPR/Cas9-mediated gene disruption of the DLGAP5 locus. This polyclonal knockout model generates a heterogeneous population of cells carrying diverse loss-of-function mutations in DLGAP5, enabling robust functional studies without clonal selection bias. It serves as a powerful tool for investigating the role of the DLGAP5 gene product, HURP, in mitotic progression and chromosome segregation, particularly within the context of colorectal cancer biology. The absence of clonal isolation ensures that the polyclonal cells retain a broader representation of the parental line’s heterogeneity, making them suitable for experiments that require a pool of genetically distinct knockouts, such as drug sensitivity profiling and synthetic lethality screens.
HT29 cells are a well-characterized human colorectal adenocarcinoma epithelial cell line originally isolated from a 44-year-old female patient. They exhibit typical features of intestinal epithelial cells, including the ability to form polarized monolayers and undergo mucin production, making them a widely used model for colorectal cancer research. These cells harbor mutations in key oncogenic pathways, such as APC and p53, representing a common genetic background observed in sporadic colorectal tumors. The HT29 model is particularly valuable for studying tumor cell proliferation, differentiation, and drug response in vitro, and the DLGAP5 knockout in this context provides a relevant system to dissect mitotic dependencies in colorectal adenocarcinoma.
DLGAP5 (Discs Large-Associated Protein 5) encodes HURP (Hepatoma Up-Regulated Protein), a microtubule-associated protein essential for mitotic spindle assembly and faithful chromosome segregation. HURP is recruited to spindle microtubules through a complex formed by Aurora A kinase (AURKA) and the scaffolding protein TPX2, where it promotes microtubule bundling and stabilization, facilitating proper kinetochore attachment and mitotic progression. Its expression is transcriptionally regulated by E2F transcription factors and FoxM1 during the cell cycle. The molecular network surrounding DLGAP5 includes interactions with Importin-beta, Dynein light chain, and the kinesin motor KIF11, and it operates within a pathway containing key mitotic regulators such as NEDD1, TACC3, and KIF2A. Disruption of DLGAP5 leads to defects in chromosome alignment, spindle pole integrity, and activation of the spindle assembly checkpoint, resulting in mitotic arrest and apoptosis.
In colorectal cancer, DLGAP5 is frequently overexpressed and correlates with poor prognosis, highlighting its role as a potential therapeutic vulnerability. The HT29 knockout model allows for the functional dissection of HURP-dependent mitotic processes within a colorectal adenocarcinoma genetic background, including the study of chromosome instability??a hallmark of colorectal cancer. Because HT29 cells express active Aurora A kinase, the DLGAP5 knockout can be employed to investigate synthetic lethal interactions with Aurora kinase inhibitors or other mitotic-targeted agents. This model also facilitates the exploration of compensatory mechanisms that may arise upon loss of spindle assembly regulation, offering insights into resistance pathways and novel drug targets in colorectal cancer.
These polyclonal DLGAP5 knockout HT29 cells support diverse assays: Western blotting and RT-qPCR for expression analysis, immunofluorescence for mitotic spindle visualization, and flow cytometry for cell cycle profiling. Proliferation, migration, and invasion assays, along with time-lapse microscopy, reveal functional consequences of DLGAP5 loss. Drug sensitivity testing with Aurora kinase inhibitors or chemotherapeutics can uncover therapeutic vulnerabilities. The polyclonal nature is ideal for functional genomic screens and synthetic lethality studies. For further information, please contact Ascent Research.
