KNTC1 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population targeting the kinetochore gene KNTC1 in the widely used HEK293T human embryonic kidney cell line. This product provides a pooled loss-of-function model, offering a heterogeneous pool of edited cells that enables robust phenotypic analysis of KNTC1 disruption on chromosome segregation and spindle assembly checkpoint signaling without clonal selection biases. The polyclonal format is ideal for studying the collective cellular response to KNTC1 ablation, avoiding artifacts that may arise from single-cell cloning.
HEK293T cells, a clonal derivative of HEK293 stably expressing SV40 large T antigen, are widely utilized for their exceptionally high transfection efficiency, rapid growth, and support for episomal replication of plasmids containing the SV40 origin. These human embryonic kidney cells are a well-characterized platform for genetic manipulation, tolerating CRISPR/Cas9 editing effectively, and are particularly suited for generating polyclonal knockout populations to investigate fundamental cellular processes such as mitosis.
KNTC1 encodes a core subunit of the NDC80 complex, which also includes NDC80/HEC1, NUF2, SPC24, and SPC25, and is essential for mediating kinetochore-microtubule attachments. Its function is tightly regulated: Aurora B kinase phosphorylates the complex to correct erroneous attachments, while E2F transcription factors drive KNTC1 expression at the G1/S transition, and CDK1-cyclin B promotes activity during mitotic entry. Downstream, the NDC80 complex recruits spindle assembly checkpoint proteins MAD1 and MAD2 to unattached kinetochores, facilitating formation of the mitotic checkpoint complex (MCC) that inhibits the anaphase-promoting complex/cyclosome until chromosomes are properly bioriented. KNTC1 also interacts with the MIS12 complex, KNL1, and Zwint-1 to maintain kinetochore integrity and signaling.
Disruption of KNTC1 abolishes NDC80 complex function, leading to defective kinetochore-microtubule attachments, persistent spindle checkpoint activation, mitotic arrest, and consequent chromosome missegregation and aneuploidy. In the HEK293T background, this polyclonal knockout serves as a powerful model for chromosomal instability (CIN), a hallmark of many cancers, and is well-suited for high-content screening to identify factors that exacerbate or rescue CIN phenotypes. It enables direct investigation of KNTC1’s role in maintaining genomic integrity, with relevance to cancers such as breast and hepatocellular carcinomas where KNTC1 dysregulation is implicated.
This knockout product supports a wide range of experimental applications, including immunofluorescence microscopy for kinetochore localization and spindle morphology, live-cell imaging to monitor mitotic progression, flow cytometry for cell cycle profiling and aneuploidy detection, and biochemical assays such as western blotting and co-immunoprecipitation to assess NDC80 complex assembly and checkpoint signaling. Colony formation assays evaluate long-term proliferative effects, while transcriptomic analysis by RNA-seq can reveal global gene expression changes. The model is particularly valuable for cancer biology, drug development targeting mitotic machinery, and synthetic lethality screens with small-molecule inhibitors. For further details or technical inquiries, please contact Ascent Research.
