The ANKIB1 Knockout HT29 Polyclonal Cells consist of a heterogeneous pool of HT29 cells with CRISPR/Cas9-mediated disruption of the ANKIB1 gene, providing a loss-of-function model for the E3 ubiquitin ligase. This polyclonal knockout population maintains editing diversity, avoiding clonal artifacts and offering a representative knockout background for functional studies.
The HT29 parental cell line, derived from a human colorectal adenocarcinoma of a 44-year-old female, is a well-established model for colon carcinoma. HT29 cells harbor mutations in APC, p53, and BRAF (V600E), making them valuable for studying oncogenic signaling, cancer progression, and drug responses. These adherent epithelial cells retain key characteristics of tumor epithelium and are routinely used in intestinal biology research.
ANKIB1 functions as an E3 ubiquitin-protein ligase within the ubiquitin-proteasome system, catalyzing ubiquitin transfer from E2 conjugating enzymes to specific substrates, targeting them for 26S proteasomal degradation. This activity regulates protein homeostasis and can modulate signaling pathways by controlling protein abundance. The pathway involves sequential action of E1, E2, and E3 enzymes, with ANKIB1 interacting with E2s, ubiquitin, and substrates. While upstream regulators of ANKIB1 are not well characterized, its downstream effects are mediated through ubiquitinated substrates destined for degradation, potentially impacting cell proliferation and survival.
Knocking out ANKIB1 in the HT29 background allows investigation of ubiquitin-dependent protein degradation in the context of colorectal cancer mutations. The combination with APC, p53, and BRAF(V600E) alterations provides a platform to study how disrupted proteostasis intersects with key oncogenic pathways, influencing tumor cell behavior and drug sensitivity. This model is particularly suited for examining the contribution of E3 ligase activity to colon carcinoma maintenance.
Research applications include identification of ANKIB1 substrates using co-immunoprecipitation and mass spectrometry, ubiquitination profiling, and proteasome activity assays. Functional studies can employ MTT proliferation assays, Annexin V apoptosis assays, colony formation, and migration/invasion analyses, complemented by RNA-seq and flow cytometry. The model also supports drug sensitivity screening to evaluate therapeutic responses. For further information, please contact Ascent Research.
