The EHD3 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from the human HT29 colorectal adenocarcinoma cell line, engineered to disrupt the EHD3 gene. This polyclonal knockout model provides a powerful tool for loss-of-function studies without clonal selection, preserving the natural heterogeneity of the host cell population. EHD3 is a key regulator of endocytic recycling, and its genetic disruption enables investigation of membrane trafficking dynamics in a cancer-relevant epithelial context.
HT29 cells are widely used as a model of intestinal epithelial biology due to their ability to form polarized monolayers, produce mucins, and undergo differentiation upon metabolic stress. These cells harbor mutations in the tumor suppressor genes APC and TP53, as well as the oncogene KRAS, making them a representative system for studying colorectal tumorigenesis. Their epithelial origin and capacity for barrier formation render them particularly suitable for examining processes such as cell adhesion, migration, and receptor trafficking.
At the molecular level, EHD3 functions downstream of Rab GTPases (e.g., Rab11) and Arf6, and is activated by receptor tyrosine kinases such as EGFR. It interacts with the endocytic machinery including EHD1, EHD4, syndapin, amphiphysin, the AP-2 complex, and F-actin to promote membrane tubulation and fission at endosomes. This activity facilitates the recycling of internalized receptors like integrin ??1 and EGFR back to the plasma membrane, thereby modulating actin cytoskeleton organization and sustaining signaling pathways that drive cell migration and invasion.
In the HT29 background, disruption of EHD3 is expected to impair the endosomal recycling of integrins and growth factor receptors, leading to altered cell surface expression and attenuated downstream signaling. This perturbation provides a physiologically relevant context to dissect the contribution of endocytic trafficking to colorectal cancer progression, particularly the metastatic cascade where enhanced recycling promotes tumor cell motility and ECM adhesion. The model thus bridges fundamental cell biology with translational oncology research.
Researchers can employ this EHD3 knockout model in a variety of assays, including Western blotting and RT-qPCR to confirm gene disruption and downstream effects, immunofluorescence and flow cytometry to assess receptor trafficking and surface expression, and migration/invasion assays to evaluate functional outcomes. Additionally, endocytosis/recycling assays, apoptosis assays, and drug sensitivity tests can be performed to explore therapeutic vulnerabilities and resistance mechanisms. For further technical details and custom inquiries, please contact Ascent Research.
