The ITSN1 Knockout HT29 Polyclonal Cells consist of a CRISPR/Cas9-edited heterogeneous population derived from the human colorectal adenocarcinoma HT29 line, with targeted disruption of the ITSN1 gene. This polyclonal pool captures diverse editing events, offering a robust loss-of-function model for studying ITSN1-dependent processes without clonal selection bias. The cells provide a physiologically relevant platform to investigate the adaptor protein ITSN1, which coordinates clathrin-mediated endocytosis with actin dynamics, and are suitable for cancer signaling, endocytic trafficking, and neurological disease research.
The host HT29 cell line was established from a 44-year-old female with colorectal adenocarcinoma and carries mutations in APC, TP53, and PIK3CA, mirroring frequent genetic lesions in colorectal tumorigenesis. These adherent epithelial cells exhibit an absorptive enterocyte-like phenotype and serve as a standard model for intestinal barrier function, colorectal cancer biology, and transepithelial drug transport studies. The oncogenic mutation background makes HT29 an ideal system for probing ITSN1 function in a disease-relevant context.
ITSN1 is a multidomain adaptor that links endocytic machinery to the actin cytoskeleton. Its EH domains bind Eps15 and Epsin, while SH3 domains recruit Dynamin to promote clathrin-coated pit fission. The DH-PH domain activates Cdc42, stimulating N-WASP/Arp2/3-mediated actin polymerization required for vesicle formation. ITSN1 is activated downstream of EGF/EGFR and Ephrin receptors, and regulated by Calmodulin and Ca2+ signals. It scaffolds Sos, connecting endocytosis to Ras/MAPK pathway activation, and interacts with Synaptojanin and PI3K-C2?? at the phosphoinositide interface.
In HT29 cells, ITSN1 knockout allows dissection of the coupling between receptor endocytosis and oncogenic signaling. EGFR is often overexpressed in colorectal cancers, and ITSN1-mediated internalization modulates receptor desensitization and downstream MAPK output. Loss of ITSN1 is anticipated to disrupt EGFR trafficking, actin organization, and cellular responses to EGF. This model thus enables analysis of how endocytic adaptor dysfunction influences cancer cell proliferation, migration, and invasion. Additionally, HT29’s enterocytic characteristics facilitate studies on transepithelial transport and drug absorption, with relevance to Alzheimer’s disease and Down syndrome where ITSN1 affects synaptic vesicle recycling and APP trafficking.
These ITSN1 knockout HT29 cells are applicable in transferrin uptake and EGFR internalization assays to quantify endocytic defects. Western blotting and RT-qPCR assess changes in Cdc42, N-WASP, and ERK phosphorylation, while actin staining visualizes cytoskeletal rearrangements. Functional assays such as wound healing and Matrigel invasion evaluate migratory and invasive properties. Co-immunoprecipitation with Eps15, Dynamin, or Cbl maps altered protein interactions. The cells also serve as a platform for intestinal drug absorption studies and neurodegenerative disease research. For further information or technical inquiries, please contact Ascent Research.
