The EHD4 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population featuring disruption of the human EHD4 gene in the HT29 colorectal adenocarcinoma cell line. This heterogeneous pool contains loss-of-function alleles generated by CRISPR/Cas9-mediated gene targeting, providing a cost-effective and rapid model for studying EHD4 function while avoiding potential clonal artifacts. The polyclonal format is ideal for experiments requiring diverse editing outcomes and robust functional readouts.
The parental HT29 cell line was established from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female and serves as a well-characterized intestinal epithelial model with an adenocarcinoma phenotype. HT29 cells are widely employed in cancer research for their ability to recapitulate key aspects of colorectal tumor biology, making them a physiologically relevant host for knockout studies focused on endocytic trafficking and signaling.
EHD4 is an Eps15 homology domain-containing protein that orchestrates endocytic recycling and membrane homeostasis. It is integral to receptor-mediated endocytosis and intracellular trafficking, particularly of EGFR and the insulin receptor. EHD4 interacts with Arf6, Rab11-FIP2, syndapin-2, and its paralog EHD1, forming complexes that regulate endosomal sorting. Upstream signals from EGF, insulin, and PI3K modulate EHD4 activity, and its function impacts downstream targets including EGFR, insulin receptor, and transferrin receptor, thereby controlling cell surface receptor levels and downstream signaling.
Disruption of EHD4 in HT29 cells likely impairs recycling of EGFR and insulin receptor, resulting in altered surface expression and aberrant activation of downstream pathways such as PI3K/AKT and MAPK. This model is particularly relevant for colorectal cancer research, where EGFR trafficking influences tumor cell proliferation and migration, and for type 2 diabetes studies examining insulin signaling cross-talk in an epithelial setting. It provides a platform to investigate how endocytic defects contribute to oncogenic and metabolic dysregulation.
This knockout cell product supports diverse experimental workflows including western blotting, RT-qPCR, and immunofluorescence for assessing protein and transcript levels. Endocytosis and recycling assays using fluorescently labeled EGF or transferrin, flow cytometry for receptor surface quantification, and functional assays such as migration and invasion studies are readily performed. Phospho-signaling analysis (e.g., AKT, ERK) and RNA-seq can further delineate pathway alterations. These cells are well suited for dissecting receptor trafficking in colorectal cancer, studying drug resistance mechanisms linked to EGFR dynamics, and exploring insulin pathway modulation. Contact Ascent Research for further technical details.
