The KCTD3 Knockout HT29 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal population of HT29 human colorectal adenocarcinoma cells in which the KCTD3 gene has been disrupted to generate a loss-of-function model. This polyclonal knockout product retains the genetic diversity of the parental cell line while providing targeted gene ablation, allowing researchers to investigate KCTD3-dependent functions without clonal selection artifacts. The pooled edited cells are suitable for applications that require robust population-level phenotypes and are immediately ready for expansion and experimental use.
The HT29 cell line is a widely used epithelial model of colorectal adenocarcinoma, originally isolated from a primary tumor of a 44-year-old female patient. These adherent cells display an epithelial morphology and retain key characteristics of intestinal epithelium, including the capacity for mucinous differentiation. HT29 cells harbor mutations in APC and TP53, among other cancer-associated genes, making them a relevant tumorigenic background for studying colorectal cancer cell signaling, proliferation, and drug responses.
KCTD3 encodes a protein containing a BTB/POZ domain that functions as a substrate adaptor for the Cullin3-RING E3 ubiquitin ligase complex, comprising Cullin3, Rbx1, and E2 enzymes, directing specific substrates for 26S proteasomal degradation. KCTD3 directly interacts with G protein ?¦? subunits, integrating G protein-coupled receptor (GPCR) signaling with the ubiquitin-proteasome system. Upon GPCR activation, G?¦? subunits may recruit KCTD3 to active signaling complexes, modulating the stability of downstream effectors. Disruption of KCTD3 expression is expected to alter ubiquitin-mediated regulation of components in the G?¦?-mediated PI3K/AKT and ERK signaling cascades, as well as potentially affect other pathways such as Wnt/??-catenin signaling, which is constitutively active in HT29 cells.
In the context of HT29 colorectal cancer cells, KCTD3 knockout provides a valuable tool for dissecting the crosstalk between ubiquitin-dependent proteolysis and GPCR signal transduction. The loss of KCTD3 may perturb the degradation of key signaling regulators, leading to aberrant activation of PI3K/AKT and ERK pathways that drive cell proliferation and survival. This model enables the examination of how KCTD3-mediated ubiquitination influences tumor cell behavior, including growth, migration, and apoptosis, in a genetic background that already features dysregulated Wnt and p53 pathways. Consequently, the KCTD3 knockout HT29 polyclonal cells serve as a physiologically relevant platform for investigating the molecular underpinnings of colorectal cancer progression.
Researchers can employ this polyclonal knockout cell population in a wide array of functional assays, including Western blot and RT-qPCR to confirm KCTD3 ablation, Sanger sequencing to verify indel formation, cell proliferation assays (MTS/MTT), and apoptosis assays (Annexin V/PI staining). The model supports transwell migration/invasion assays, co-immunoprecipitation with G?¦? and Cullin3, ubiquitination analyses, and phospho-signaling profiling of AKT and ERK. Additional applications include drug sensitivity screening with G protein modulators and RNA-seq transcriptome profiling. For further details or to discuss how this knockout product can accelerate your research, please contact Ascent Research.
