The EFNA5 Knockout HT29 Polyclonal Cells constitute a heterogeneous population of colorectal adenocarcinoma cells engineered via CRISPR/Cas9-mediated disruption of the EFNA5 gene locus. This polyclonal knockout model eliminates functional ephrin-A5 protein expression across a mixed pool of edited cells, providing a powerful tool for studying loss-of-function effects in a genetically diverse background. The product is supplied as a ready-to-use polyclonal cell population, circumventing the need for single-cell cloning while enabling robust functional assays.
The HT-29 host cell line, established from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female, exhibits epithelial morphology and serves as a well-characterized model for intestinal epithelial biology and colorectal cancer research. HT-29 cells retain key features of transformed intestinal epithelia, including the capacity for differentiation and responsiveness to various signaling cues, making them a suitable platform for investigating tumor suppressor gene function and oncogenic pathways.
EFNA5 encodes ephrin-A5, a glycosylphosphatidylinositol (GPI)-anchored ligand that engages EphA receptor tyrosine kinases, particularly EPHA2, EPHA3, EPHA4, and EPHA7. Ligation triggers bidirectional signaling: forward signaling through the Eph receptors and reverse signaling through the ephrin ligand, often mediated by SRC family kinases and FYN. Downstream effectors include Rho family GTPases RHOA, RAC1, and CDC42, as well as the PI3K-AKT and MAPK pathways, involving AKT1 and MAPK1. EFNA5 signaling regulates cytoskeletal dynamics via ROCK1, LIMK1, and CFL1, and modulates focal adhesion kinase (PTK2) activity. Upstream regulators include TP53, NFKB1, CTNNB1, and DNA methylation, placing EFNA5 at the intersection of tumor suppressive and developmental signaling networks.
In the colorectal cancer context, EFNA5 functions as a tumor suppressor, and its loss is associated with enhanced cell migration and invasion. In HT-29 cells, CRISPR/Cas9-mediated knockout of EFNA5 is predicted to relieve contact-dependent repulsion, promoting collective cell migration and invasive behavior. This model enables dissection of EFNA5’s role in modulating adhesion and repulsion through EPHA2 and EPHA4 interactions, as well as downstream activation of SRC and RHOA. The polyclonal nature of the knockout population allows assessment of heterogeneous cellular responses, recapitulating aspects of tumor heterogeneity.
This knockout product is ideally suited for a range of functional assays, including wound healing and Transwell invasion assays to quantify migratory and invasive potential, MTT assays for proliferation, and immunoblotting or RT-qPCR to confirm loss of ephrin-A5 and assess pathway alterations such as phospho-AKT levels. It provides a versatile platform for studying Eph-ephrin signaling dynamics, drug resistance mechanisms, and therapeutic target validation in colorectal cancer. For further technical specifications and support, please contact Ascent Research.
