CASP8 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from HEK293T cells, designed for studying caspase-8 function. The product offers a heterogeneous pool of cells with disrupted CASP8 gene, enabling loss-of-function studies without clonal selection. This polyclonal format reduces clone-specific artifacts and provides a robust model for apoptosis and necroptosis research. It serves as a foundational tool for investigating extrinsic cell death signaling.
The HEK293T cell line is a human embryonic kidney epithelial line transformed with adenovirus type 5 DNA and expressing SV40 large T-antigen, which facilitates episomal replication of plasmids. These cells are widely used for protein expression, virus production, and gene editing due to high transfectability and rapid growth. In the context of caspase-8 knockout, HEK293T provides a tractable system to interrogate death receptor pathways while maintaining key signaling components. The well-characterized nature ensures reproducibility and compatibility with standard assays.
Caspase-8 is an initiator caspase that plays a pivotal role in extrinsic apoptosis, activated upon ligand binding to death receptors such as FAS, TNFRSF10A/B, and TNFRSF1A. In the canonical pathway, procaspase-8 is recruited to the death-inducing signaling complex (DISC) via the adaptor FADD, leading to dimerization and autocatalytic processing. Active caspase-8 then cleaves downstream executioner caspases-3 and -7 to initiate apoptosis, or cleaves BID to engage the mitochondrial pathway through cytochrome c release and APAF1/caspase-9 apoptosome formation. Beyond apoptosis, caspase-8 regulates necroptosis by interacting with RIPK1 and RIPK3, and modulates inflammatory signaling via NF-kappaB. It also interacts with FLIP, TRAF2, and caspase-10.
In HEK293T cells, disrupting CASP8 generates a loss-of-function model to dissect death receptor signaling. These polyclonal knockout cells enable studying resistance to death ligand-induced apoptosis by abolishing the major initiator caspase. They also permit investigation of caspase-8-independent necroptosis under RIPK1-driven conditions. The absence of caspase-8 may affect NF-kappaB activation and autophagic responses, providing a platform to explore cross-talk between cell death modalities. Given the HEK293T background, the knockout cells retain utility for transient expression studies, allowing rescue experiments with mutant caspase-8 constructs.
Researchers can employ these cells in apoptosis assays using flow cytometry for Annexin V/propidium iodide staining, complemented by western blotting for cleaved caspase-3 to assess pathway activation downstream of death receptors. Co-immunoprecipitation of DISC components enables dissection of caspase-8 interactions with FADD, FLIP, and RIPK1. For necroptosis studies, treatment with TNF-alpha in the presence of caspase inhibitors reveals RIPK3-dependent cell death. Drug screening campaigns can utilize these cells to identify compounds that restore apoptosis sensitivity. The polyclonal population also supports CRISPR screens to uncover synthetic lethal interactions or novel regulators of cell death. For further information, please contact Ascent Research.
