The DYNC2H1 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout population targeting the DYNC2H1 gene. The product consists of a bulk pool of HEK293T cells harboring heterogeneous gene disruptions, thereby avoiding the selective pressures and artifacts associated with monoclonal lines. This polyclonal design provides a robust and reproducible loss-of-function model for dissecting DYNC2H1-related mechanisms.
HEK293T cells are a derivative of HEK293 that stably express the SV40 large T-antigen, enabling episomal replication of SV40 origin-containing plasmids. This feature supports high-level protein expression and makes the line exceptionally amenable to transfection, viral production, and gene editing. The adherent, fast-growing cells are a standard model in cell biology. Under serum starvation, HEK293T cells form primary cilia, establishing them as a relevant platform for ciliogenesis and Hedgehog pathway studies.
DYNC2H1 encodes the heavy chain of cytoplasmic dynein-2, the motor for retrograde intraflagellar transport (IFT) in primary cilia. The dynein-2 complex (including DYNC2LI1, WDR34, WDR60) cooperates with IFT-A and IFT-B. Retrograde IFT is vital for cilia maintenance and Hedgehog pathway function. Ciliogenesis inducers and transcription factors (RFX family, FOXJ1) regulate DYNC2H1. Its disruption halts retrograde trafficking, blocks ciliogenesis, and impairs GLI transcription factor processing??reducing GLI3 repressor formation and GLI activator, leading to suppressed expression of SHH targets (PTCH1, GLI1). This occurs downstream of PTCH1/SMO and upstream of SUFU.
In HEK293T, the DYNC2H1 knockout provides a tractable system for dissecting retrograde IFT and Hedgehog signaling. The cells’ high transfectability facilitates rescue experiments with wild-type or mutant DYNC2H1. Serum-starved control cells form cilia and activate Hedgehog targets, while knockout cells fail ciliogenesis and show attenuated responses. The model is ideal for studying GLI activator/repressor dynamics and ciliary SMO trafficking, with the polyclonal nature ensuring phenotypes are not clonal artifacts.
This knockout population enables diverse assays: immunofluorescence for ciliary markers (acetylated tubulin, ARL13B), Western blotting for GLI3 processing, and RT-qPCR for target genes (PTCH1, GLI1). Live-cell imaging can visualize IFT defects, while pharmacological challenge (SMO agonists) maps pathway epistasis. The cells serve as a model for ciliopathies like short-rib thoracic dysplasia and Jeune asphyxiating thoracic dystrophy, and for screening ciliary modulators. For further information, contact Ascent Research.
