This product comprises a CRISPR/Cas9-edited polyclonal HT29 cell population carrying targeted disruption of the ITCH gene, providing a loss-of-function model for the ITCH E3 ubiquitin ligase. HT29 cells were engineered using CRISPR/Cas9 to introduce gene disruptions across the ITCH locus, yielding a heterogeneous knockout polyclonal pool suitable for functional studies. The polyclonal format maintains genetic diversity while silencing ITCH function, enabling robust analysis of ITCH-dependent phenotypes in a colorectal adenocarcinoma background.
The HT29 cell line was originally derived from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female. These adherent, epithelial-like cells retain the capacity to differentiate into enterocyte-like and mucus-producing phenotypes under appropriate culture conditions, making them a versatile model for intestinal epithelial biology. HT29 cells harbor mutations in key oncogenic drivers such as APC, TP53, and BRAF, reflecting genetic alterations commonly observed in colorectal cancer. Consequently, HT29 is widely employed to investigate colorectal cancer pathogenesis, drug responses, and signaling pathway dynamics in a cellular context that recapitulates clinically relevant molecular features.
ITCH encodes a HECT-type E3 ubiquitin ligase that catalyzes polyubiquitination of key signaling proteins. Activated by JNK phosphorylation and adaptors like NDFIP1, it targets transcription factors c-Jun and p73, the Hippo kinase LATS1, and Notch1. In the Hippo pathway, ITCH promotes YAP/TAZ degradation via LATS1/2, suppressing proliferation. ITCH-mediated turnover of c-Jun modulates AP-1 activity, while Notch1 ubiquitination alters receptor stability. These functions integrate JNK, Hippo, and Notch pathways, regulating apoptosis, immune responses, and cellular homeostasis.
In HT29 colorectal cancer cells, ITCH-mediated ubiquitination influences key processes tied to intestinal tumor biology. Disruption of ITCH in this model allows investigation of how loss of this ligase alters JNK-mediated AP-1 transcriptional activity, Hippo-dependent YAP/TAZ stability, and Notch1 receptor turnover. Additionally, ITCH regulates the stability of p73 and p63, impacting DNA damage response and apoptotic balance. Given its role in degrading both tumor suppressors and oncoproteins, ITCH knockout in HT29 enables dissection of context-dependent signaling outcomes. This model is particularly relevant for elucidating mechanisms of resistance to chemotherapeutic agents, as ITCH has been implicated in modulating sensitivity to drugs like fluorouracil and oxaliplatin.
Key applications include functional analysis of E3 ligase activity in colorectal cancer, mapping ITCH-substrate interactions by ubiquitination assays and co-immunoprecipitation, and studying JNK/Hippo crosstalk. The cells are suitable for high-throughput screening of ITCH modulators, drug resistance studies (IC50 determination), and phenotypic assays for proliferation, apoptosis, and colony formation. Gene and protein expression analyses via RT-qPCR and Western blotting validate pathway effects. This ITCH knockout model provides a versatile tool for preclinical research. For further information, contact Ascent Research.
