The HERC1 Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human colorectal adenocarcinoma HT29 cell line. This product features targeted disruption of the HERC1 gene. The polyclonal format provides a heterogeneous pool of cells with loss-of-function mutations, suitable for studying HERC1-dependent signaling without clonal selection artifacts.
The HT29 host cell line is a well-established epithelial model originating from human colorectal adenocarcinoma. These adherent cells express adenocarcinoma markers and are extensively employed in research on proliferation, differentiation, metastasis, and drug response. Their genetic landscape mirrors common colorectal cancer mutations, making them ideal for investigating mTOR and MAPK/ERK pathways, which are frequently aberrantly activated in colon tumors.
HERC1 functions as an E3 ubiquitin ligase that directly ubiquitinates TSC2, promoting its proteasomal degradation. This relieves TSC2-mediated inhibition of rheb, activating mTORC1 and phosphorylation of downstream effectors S6K and 4E-BP1, which drive protein synthesis and cell growth. Upstream, ERK (MAPK1/3), activated by growth factor signaling, phosphorylates HERC1 to modulate its activity. Additionally, HERC1 interacts with clathrin heavy chain and ARF6, facilitating clathrin-mediated endocytosis. Thus, HERC1 integrates growth factor signals with mTORC1 and membrane trafficking.
In HT29 colorectal adenocarcinoma cells, HERC1 knockout stabilizes TSC2, leading to mTORC1 inhibition and reduced proliferation, offering a relevant model for colorectal cancer where mTOR hyperactivity drives tumor growth and drug resistance. The interplay between mTOR and MAPK/ERK pathways can be dissected. Given the oncogenic role of mTOR, this model serves as a powerful tool to elucidate consequences of mTORC1 downregulation on tumor cell behavior. Although HERC1 mutations are linked to neurodevelopmental disorders such as intellectual disability, macrocephaly, and autism, this system provides a foundation for studying HERC1 loss in a cellular context.
These polyclonal knockout cells enable investigation of mTOR signaling by quantifying phospho-S6K and phospho-4E-BP1 levels via western blotting, assessing proliferation via MTT and clonogenic assays, and monitoring autophagic flux. Co-immunoprecipitation and ubiquitination assays reveal TSC2 ubiquitination status; immunofluorescence visualizes membrane trafficking defects. The model is suitable for colorectal cancer drug resistance studies and exploring mTOR/MAPK crosstalk. For further details and technical support, please contact Ascent Research.
