The CBS Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-engineered polyclonal population designed to eliminate CBS gene expression through targeted gene disruption. This loss-of-function model enables the study of cystathionine beta-synthase (CBS) function in the transsulfuration pathway and hydrogen sulfide (H2S) biosynthesis. By generating a diverse pool of edited cells, this product provides a robust system for investigating metabolic and signaling outcomes without clonal selection biases.
The parental HEK293T cell line is a widely utilized human embryonic kidney epithelial model derived from HEK293 cells and transformed with adenovirus 5 DNA. These cells stably express the SV40 large T antigen, which promotes high-level episomal replication of plasmids containing the SV40 origin, resulting in exceptional transfection efficiency and protein expression. HEK293T cells are thus an ideal host for gene knockout studies, offering technical advantages for downstream molecular and biochemical analyses.
CBS encodes a pyridoxal 5??-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine, a critical step in the transsulfuration pathway linking methionine cycle to cysteine metabolism. CBS activity is allosterically stimulated by S-adenosylmethionine (SAM) and requires a heme cofactor for proper folding and function. It is regulated by cellular redox status and hypoxia. The enzyme also generates H2S, a gaseous signaling molecule. Cystathionine is subsequently converted to cysteine by cystathionine gamma-lyase (CTH), supplying precursor for glutathione synthesis and additional H2S production. Thus, CBS sits at a metabolic junction, influencing homocysteine clearance, cysteine availability, antioxidant capacity, and gasotransmitter signaling.
In HEK293T cells, disruption of CBS abrogates transsulfuration, leading to accumulation of homocysteine and decreased levels of cystathionine, cysteine, glutathione, and H2S. This metabolic shift induces oxidative stress and impairs redox signaling, mirroring aspects of homocystinuria and hyperhomocysteinemia. The model is particularly valuable in this epithelial background for dissecting cellular responses to homocysteine toxicity, hydrogen sulfide-mediated pathways, and sulfur amino acid homeostasis. The high transfectability of HEK293T cells also facilitates complementation studies with wild-type or mutant CBS constructs to validate phenotypic rescue.
Researchers can employ these polyclonal knockout cells in a variety of investigative contexts, including the study of homocysteine metabolism, H2S signaling, and redox biology. Representative assays include Western blotting to confirm loss of CBS protein, CBS enzyme activity measurements, HPLC-based homocysteine quantification, fluorescent probe detection of H2S, glutathione level assessment, and qRT-PCR analysis of pathway gene expression. This knockout model is well-suited for disease modeling of homocystinuria, cardiovascular and neurological disorders, and for screening small molecules that modulate transsulfuration. For further technical information, please contact Ascent Research.
