The CASP3 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population featuring targeted disruption of the human CASP3 gene. This model eliminates functional caspase-3, the principal executioner protease in apoptotic cell death, providing a powerful tool for dissecting the molecular control of apoptosis. As a polyclonal pool, the cells retain genetic heterogeneity, making them well-suited for population-level functional assays and for studies where clonal variation is not a limiting factor.
The parent HEK293T line is an immortalized human embryonic kidney epithelial cell line that constitutively expresses the SV40 large T antigen. This feature enables high-copy episomal replication of plasmids bearing the SV40 origin, thereby conferring exceptionally high transfection efficiency and robust recombinant protein expression. HEK293T cells are a workhorse in biomedical research, routinely used for lentiviral packaging, transient overexpression, and gene editing experiments, and they provide a reliable and experimentally tractable background for knockout generation.
The CASP3 gene product, caspase-3, is synthesized as an inactive zymogen and becomes activated by proteolytic cleavage mediated by initiator caspases such as caspase-8 and caspase-9. Caspase-8 is activated within the death-inducing signaling complex following death receptor ligation (FasL/Fas, TNF/TNFR), while caspase-9 is activated by Apaf-1 in the apoptosome after cytochrome c release. Active caspase-3 cleaves substrates including PARP, DFF40/ICAD, gelsolin, fodrin, and lamin A, executing apoptotic cell death. Its activity is regulated by inhibitor-of-apoptosis proteins like XIAP and is downstream of Bcl-2 family-controlled mitochondrial permeabilization. Additionally, granzyme B from cytotoxic lymphocytes can directly process caspase-3.
Disruption of CASP3 in HEK293T cells ablates the execution step of both the extrinsic and intrinsic apoptotic pathways, creating a clean loss-of-function model. This knockout enables researchers to study caspase-3-independent cell death modalities, including necroptosis and ferroptosis, and to explore the non-apoptotic functions of caspase-3 that are increasingly recognized in processes such as cell differentiation, tissue remodeling, and inflammatory signaling. In cancer research, these cells are instrumental for modeling chemoresistance driven by apoptosis evasion. In neurodegeneration, they facilitate the investigation of alternative cell death mechanisms underlying neuronal loss.
Representative applications include apoptosis monitoring via Western blot detection of cleaved PARP and caspase-3, flow cytometry with Annexin V/propidium iodide staining, cell viability assays (MTT, CTG), and fluorometric or luminescent caspase-3 activity measurements. The model is ideally suited for high-throughput screening of small molecules that reactivate apoptosis or inhibit caspase-3 in non-apoptotic contexts. It also supports structure-function studies by reconstitution with mutant caspase-3 variants and interrogation of the p53 signaling pathway. For comprehensive product details, validation reports, and ordering assistance, please reach out to Ascent Research.
