The ARAF Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to eliminate ARAF protein expression in the HCT 116 human colorectal carcinoma cell line. This product comprises a pool of cells with heterogeneous CRISPR/Cas9-mediated disruptions, ensuring robust gene knockout across the population. As polyclonal cells, they offer a convenient and physiologically relevant model for studying ARAF function without the bottlenecks of single-cell cloning, making them suitable for high-throughput applications and detailed mechanistic studies.
HCT 116 is a well-established epithelial cell line derived from a patient with colorectal carcinoma. It exhibits microsatellite instability and carries an oncogenic KRAS G13D mutation, both hallmarks of aggressive colorectal cancers. These cells are adherent, proliferate rapidly, and are extensively characterized in oncology research. The KRAS mutation drives constitutive MAPK pathway activation, positioning HCT 116 as an ideal host for dissecting RAF isoform-specific signaling dependencies.
The ARAF gene encodes a serine/threonine kinase that functions within the RAS-RAF-MEK-ERK signaling cascade. Upon activation by RAS GTPases such as KRAS and NRAS, ARAF phosphorylates and activates MEK1 and MEK2, leading to ERK1/2 phosphorylation. Upstream inputs include receptor tyrosine kinases (e.g., EGFR, FGFR, PDGFR), SRC family kinases, and PKA. ARAF forms complexes with 14-3-3 proteins, KSR1, and HSP90, which regulate its stability and activity. Downstream, activated ERK phosphorylates transcription factors like ELK1, promoting expression of c-FOS and c-MYC to control proliferation and survival. Relative to BRAF and CRAF, ARAF has lower intrinsic kinase activity and is hypothesized to modulate pathway output or scaffold protein interactions.
In the KRAS-mutant HCT 116 context, ARAF knockout disrupts the MAPK signaling equilibrium, potentially attenuating ERK activation despite the presence of oncogenic KRAS. This model allows researchers to distinguish ARAF-specific functions from those of BRAF and CRAF, and to explore how cancer cells compensate for RAF isoform loss. The resultant phenotypes may include slowed cell growth, G1/S cell cycle arrest, or increased apoptosis, providing insights into ARAF??s contribution to colorectal cancer cell maintenance and its potential as a therapeutic target.
These polyclonal knockout cells are compatible with a wide array of experimental techniques. Western blotting for phospho-ERK provides direct readout of pathway inhibition; RT-qPCR quantifies immediate early gene transcription. Proliferation, colony formation, flow cytometry for cell cycle and apoptosis enable functional phenotyping. Drug sensitivity testing with MAPK pathway inhibitors reveals resistance mechanisms. RNA-sequencing and co-immunoprecipitation map ARAF-dependent transcriptional and protein interaction networks. This model supports functional genomics, synthetic lethality screens, and drug discovery. For further details, contact Ascent Research.
