The CBY1 Knockout HEK293T Polyclonal Cells product provides a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the CBY1 gene, enabling loss-of-function studies in a widely used human embryonic kidney epithelial background. This polyclonal knockout model is generated by introducing Cas9 and guide RNAs into HEK293T cells, resulting in a heterogeneous pool of edited alleles, without clonal selection. The product is designed for researchers investigating the negative regulatory roles of CBY1 in Wnt/??-catenin signaling and ciliogenesis, and is delivered as a ready-to-use polyclonal population, avoiding artifacts associated with single-cell cloning while preserving genetic diversity representative of the bulk-edited population.
Homo sapiens HEK293T cells are an immortalized epithelial line derived from human embryonic kidney, transformed with adenovirus 5 DNA and constitutively expressing the SV40 large T antigen. These features confer robust proliferation, high transfection efficiency, and exceptional capacity for recombinant protein expression and viral production, making them a workhorse model for cell biology, biochemistry, and drug discovery. The renal epithelial origin and stable karyotype further support investigations into signaling pathways, cell cycle regulation, and crosstalk between proliferation and differentiation programs. The presence of large T antigen enables episomal replication of plasmids containing the SV40 origin, a property widely exploited in lentivirus and AAV production workflows, while the epithelial phenotype provides a relevant context for studying polarized cell functions and membrane trafficking.
CBY1 encodes a conserved protein that functions as a critical negative regulator of canonical Wnt/??-catenin signaling by directly binding ??-catenin (CTNNB1) and inhibiting its association with TCF/LEF transcription factors, thereby repressing the transcription of Wnt target genes such as MYC, CCND1, and AXIN2. The interaction between CBY1 and ??-catenin is modulated by upstream components including AKT and Wnt ligand?Creceptor complexes (Frizzled/LRP6), and is antagonized by 14-3-3 proteins (YWHAB) that sequester phosphorylated CBY1. Independent of its Wnt-regulatory role, CBY1 localizes to the basal body via interaction with PCM1 and is essential for primary cilium assembly, where it coordinates with ciliogenic factors like IFT88, CEP164, and ARL13B. Additional interactions with CARM1 link CBY1 to chromatin remodeling and transcriptional co-activation, highlighting its multifunctional nature at the crossroads of signal transduction and cytoskeletal organization.
In HEK293T cells, which possess an active autocrine Wnt signaling loop and can be experimentally induced to form primary cilia under serum starvation, CBY1 knockout provides a physiologically relevant model to dissect the interplay between ??-catenin?Cdependent transcription and ciliogenesis. Disruption of CBY1 is expected to relieve inhibition on ??-catenin/TCF-mediated transcription, leading to elevated expression of pro-proliferative and Wnt-responsive genes, while potentially compromising ciliary formation and signaling competence. These phenotypic consequences are ideally studied in this well-characterized epithelial background, where the strong expression of the SV40 large T antigen does not interfere with canonical Wnt pathway readouts, and where high transfection efficiency facilitates reconstitution experiments. The polyclonal nature of the edited population minimizes clonal drift and allows direct comparison with parental HEK293T cells cultured under identical conditions.
This polyclonal CBY1 knockout cell model is a versatile tool for investigating Wnt/??-catenin pathway dynamics, ciliary protein trafficking, and the molecular etiology of ciliopathies and cancers where CBY1 is deregulated. Representative applications include luciferase reporter assays using SuperTOPFlash constructs to quantify TCF/LEF transcriptional activity, western blotting and RT-qPCR to monitor changes in ??-catenin, MYC, and CCND1 levels, co-immunoprecipitation to assess ??-catenin?CCBY1 complex formation, and immunofluorescence microscopy to visualize ciliary markers such as ARL13B and acetylated tubulin. The cell population is also amenable to cell cycle analysis by flow cytometry and to chemical or genetic perturbations targeting upstream kinases like AKT or Wnt receptors. For detailed technical support or customized protocols, contact Ascent Research.
