The C2orf68 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population with targeted disruption of the C2orf68 (CFAP68) gene. This model, generated by transient Cas9 and guide RNA expression, contains a heterogeneous mix of edited alleles, avoiding clonal selection artifacts. The polyclonal format provides a genetically diverse background that better represents the complexity of gene editing outcomes and minimizes the risk of clonal adaptation, making it ideal for functional studies of ciliary biology and ciliopathy-related pathways.
HEK293T cells, originally derived from human embryonic kidney, stably express the SV40 large T antigen, which enables episomal replication of plasmids containing the SV40 origin and confers exceptionally high transfection efficiency. These epithelial-like cells are a mainstay for recombinant protein production and retrovirus generation, and they are widely employed in loss-of-function studies due to their tractability. Under serum starvation conditions, HEK293T cells can form primary cilia, thus providing a convenient model system for investigating ciliogenesis despite the absence of motile cilia.
C2orf68 encodes CFAP68, a coiled-coil domain-containing protein that is transcriptionally activated by FOXJ1 and RFX transcription factors. CFAP68 interacts with axonemal dynein arms and tubulin, placing it at the core of axoneme assembly and ciliary motility. It functions downstream of the intraflagellar transport protein IFT88 and upstream of the outer dynein arm heavy chain DNAH5, contributing to the regulation of ciliary beat frequency and fluid flow. Genetic disruption of C2orf68 is expected to impair both primary cilia function and ciliary protein trafficking, establishing a direct link to primary ciliary dyskinesia and related ciliopathies. The protein network also includes SPAG6, and its activity is modulated by Notch and Wnt signaling pathways.
In HEK293T cells, which form primary cilia but lack motile cilia, C2orf68 knockout enables dissection of its role in ciliary biogenesis and intraflagellar trafficking. The polyclonal nature of this knockout population reduces artifacts from clonal selection and provides a genetically diverse background, making it ideal for studying non-motile ciliary functions such as transition zone integrity and ciliary signaling. Furthermore, upon expression of FOXJ1, HEK293T cells can be induced toward a motile cilia phenotype, extending the utility of this model to studies of ciliary beat frequency and fluid flow.
Typical research applications include immunofluorescence staining of ciliary markers such as acetylated ??-tubulin and Arl13b, western blotting of axonemal proteins including DNAH5 and SPAG6, and RT-qPCR profiling of ciliogenesis-related genes. Flow cytometry can be used to quantify ciliary protein expression, and high-speed videomicroscopy enables measurement of ciliary beat frequency after motile cilia induction. This model also supports functional rescue experiments, drug screening for ciliopathies, and synthetic genetic interaction studies, and can serve as a control for tissue-specific C2orf68 knockouts in organoid or animal models. For comprehensive product information and technical assistance, please contact Ascent Research.
