The CASC3 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the CASC3 gene, a core exon junction complex (EJC) component. This model facilitates broad investigation of CASC3-dependent nonsense-mediated mRNA decay (NMD) and mRNA localization without clonal selection artifacts, offering a physiologically relevant heterogeneous cell pool for functional genomics studies.
HEK293T is a human embryonic kidney epithelial cell line transformed with SV40 large T antigen, enabling high-efficiency transfection and episomal plasmid replication. These cells are extensively employed for protein expression, lentiviral packaging, and CRISPR-based engineering due to their robust growth kinetics, well-defined transcriptome, and ease of genetic manipulation, providing an optimal host context for interrogating post-transcriptional regulation.
CASC3 is a key EJC nucleator, deposited ~20?C24 nucleotides upstream of exon?Cexon junctions during splicing. It forms a stable core with eIF4A3, MAGOH, and Y14, and subsequently recruits UPF1, UPF2, and UPF3B to initiate NMD of transcripts containing premature termination codons. In addition, CASC3 promotes cytoplasmic mRNA localization??such as that of ACTB??and influences translation. Its activity is regulated by spliceosome assembly and transcription factors like MYC, and it interacts with NXF1 and RNPS1 to coordinate mRNA export and surveillance pathways.
Disruption of CASC3 in HEK293T cells impairs EJC integrity and abolishes UPF-mediated NMD, resulting in stabilization of aberrant mRNAs that would otherwise be degraded. This knockout model is valuable for dissecting post-transcriptional gene regulation mechanisms dysregulated in breast, gastric, and colorectal cancers, as well as neurodevelopmental disorders associated with intellectual disability. It allows assessment of CASC3’s impact on cell proliferation, migration, and survival in a tractable epithelial system.
Researchers can utilize these polyclonal knockout cells in diverse assays: western blotting and RT-qPCR confirm target depletion and NMD substrate accumulation; RNA-seq identifies transcriptome-wide changes; NMD reporter assays quantify pathway activity; co-immunoprecipitation and immunofluorescence visualize CASC3 interactions with UPF1 or eIF4A3; and flow cytometry, proliferation, and migration assays evaluate phenotypic outcomes. These applications support investigations into mRNA surveillance, cancer research, and therapeutic target validation. For additional product information, please contact Ascent Research.
