The ASPH Knockout HEK293T Polyclonal Cells product provides a CRISPR/Cas9-edited polyclonal cell population harboring targeted disruption of the human ASPH gene. This loss-of-function model is designed to abrogate aspartate ??-hydroxylase activity, enabling systematic investigation of ASPH-dependent signaling pathways in a tractable human epithelial system. The polyclonal format avoids clonal selection biases, offering a heterogeneous knockout pool suitable for functional genomics, pathway dissection, and drug target validation studies.
HEK293T cells are an adherent human embryonic kidney epithelial line immortalized by stable expression of the SV40 large T antigen. Derived from the parental HEK293 lineage, they exhibit robust proliferation, high transfection efficiency, and broad utility for recombinant protein expression, viral packaging, and genome editing applications. The epithelial origin and endogenous expression of relevant signaling machinery make them a well-suited host for studying ASPH-mediated processes such as cell adhesion, migration, and epithelial-mesenchymal transition (EMT).
ASPH encodes a 2-oxoglutarate- and Fe2+-dependent dioxygenase that catalyzes post-translational hydroxylation of aspartate residues within EGF-like domains of Notch receptors and ligands, notably NOTCH1 and JAG1. This modification is critical for receptor-ligand interaction and subsequent activation of the Notch signaling cascade, which transduces signals through the ??-secretase complex, the transcription factor RBPJ, and downstream effectors such as HES1. ASPH is transcriptionally induced by hypoxia via HIF-1??, linking oxygen sensing to Notch pathway potentiation. Knockout of ASPH abolishes this hydroxylation, leading to impaired Notch activation and downregulation of mesenchymal markers including SNAI1, vimentin, MMP2, and MMP9, thereby attenuating cell migration and invasion.
In the HEK293T context, ASPH disruption creates a relevant model for exploring oncogenic mechanisms, as ASPH is frequently upregulated in carcinomas??including hepatocellular carcinoma, cholangiocarcinoma, and pancreatic cancer??and drives tumor progression and metastasis. The epithelial background allows direct assessment of cell-autonomous effects on Notch-mediated proliferation, adhesion, and EMT without confounding stromal interactions. Polyclonal cells capture the spectrum of editing outcomes, enabling robust statistical comparisons and functional screening of pathway perturbations under normoxic and hypoxic conditions.
Typical applications include Western blotting and RT-qPCR to quantify Notch pathway components and EMT markers, immunofluorescence for localization studies, wound healing and Transwell invasion assays to measure migration and invasiveness, Notch reporter assays for signaling activity, and colony formation or proliferation assays to evaluate tumorigenic potential. These cells are also valuable for HIF-1???Chypoxia response studies and for validating ASPH as a therapeutic target. For further technical information or assistance, please contact Ascent Research.
