The EHD4 Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for the targeted disruption of the EHD4 gene in the HCT 116 human colorectal carcinoma cell line. This polyclonal pool contains a heterogeneous mix of cells harboring diverse gene-editing events across the EHD4 locus, generated using CRISPR/Cas9-mediated non-homologous end joining (NHEJ). Unlike a monoclonal cell line, this format preserves genetic diversity, making it suitable for studies requiring population-level analysis of EHD4 loss-of-function. It provides a robust tool for investigating endocytic trafficking and receptor recycling pathways in a colorectal cancer context without the selection bias inherent to single-cell clones.
The HCT 116 cell line is a well-characterized epithelial colorectal adenocarcinoma model isolated from a human colon tumor. It harbors an activating KRAS G13D mutation, is deficient in the DNA mismatch repair protein MLH1, and exhibits microsatellite instability-high (MSI-H) status, while retaining wild-type p53 function. These genetic features render HCT 116 cells particularly relevant for studying the molecular mechanisms of colorectal cancer, including dysregulated growth factor signaling and tumor progression. The endogenous oncogenic background allows researchers to evaluate the impact of EHD4 disruption within a clinically pertinent framework of colon carcinogenesis.
EHD4 (Eps15 Homology Domain-containing protein 4) is an ATPase that orchestrates endocytic recycling by regulating membrane fission and fusion events, enabling the return of internalized receptors from endosomes to the cell surface. Acting downstream of EGF stimulation, EHD4 functions in concert with Rab11, Rab5, and interacting partners Syndapin and Amphiphysin. Its role in recycling key receptors such as EGFR and integrins directly impacts the amplitude and duration of MAPK and AKT signaling pathways. Consequently, EHD4 knockout in HCT 116 cells disrupts receptor recycling, leading to altered surface receptor levels and aberrant downstream signaling.
In HCT 116 cells, EHD4 knockout is expected to impair recycling of oncogenic drivers and adhesion molecules, attenuating proliferative and migratory signals. Combined with the activating KRAS G13D mutation, EHD4 loss may uncover synthetic vulnerabilities or resistance mechanisms, offering insights into therapeutic strategies. The MLH1-deficient, MSI-H background further aligns this model with a subset of colorectal cancers, providing a platform to study the intersection of membrane trafficking defects and genomic instability.
This polyclonal knockout model supports diverse applications, including endocytic recycling kinetics, receptor trafficking, cell migration, and drug response profiling. Users can employ Western blotting and RT-qPCR for EHD4 disruption confirmation, RNA-seq for transcriptomic analysis, flow cytometry for receptor surface quantification, and immunofluorescence for localization studies. Endocytosis and recycling assays, phospho-signaling analysis, and migration/invasion assays provide functional validation, while drug sensitivity assays screen for altered therapeutic responses. For further information, please contact Ascent Research.
