The DYNC2H1 Knockout 786-O Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population engineered for disruption of the DYNC2H1 gene in the human 786-O clear cell renal cell carcinoma (ccRCC) background. This pooled knockout model is generated using CRISPR/Cas9-mediated gene disruption, providing a heterogeneous population of cells with targeted loss-of-function mutations in DYNC2H1.
The 786-O cell line is a well-characterized model of VHL-deficient clear cell renal cell carcinoma, harboring a von Hippel-Lindau (VHL) tumor suppressor gene mutation. VHL loss leads to constitutive stabilization of hypoxia-inducible factors (HIFs), driving a pseudohypoxic state and contributing to tumorigenesis, angiogenesis, and metabolic reprogramming. This genetic background is particularly relevant for studying crosstalk between hypoxia signaling and cilia-dependent pathways, as cilia loss has been implicated in VHL-mutant ccRCC progression.
DYNC2H1 encodes cytoplasmic dynein 2 heavy chain, a core subunit of the dynein-2 motor complex essential for retrograde intraflagellar transport (IFT) within primary cilia. Retrograde IFT is required for the return of IFT particles, turnover of ciliary signaling components, and ultimately for ciliary maintenance and Hedgehog (Hh) signal transduction. DYNC2H1 functions downstream of anterograde IFT-B complexes (e.g., IFT88, IFT172) and interacts with dynein-2 accessory subunits including DYNC2LI1, WDR34, WDR60, and TCTEX1D2. Disruption of DYNC2H1 impairs retrograde transport critical for Gli transcription factor processing; in the absence of functional DYNC2H1, Gli activator forms fail to be generated, preventing expression of Hh target genes such as GLI1 and PTCH1. This knockout uncouples the Hedgehog pathway from its ciliary compartment, blocking signal transmission downstream of Smoothened (SMO) and SUFU.
In VHL-deficient 786-O cells, the interplay between hypoxia-driven pathways and ciliary signaling is an area of active investigation, with evidence suggesting that VHL loss may suppress ciliogenesis in a HIF-dependent manner. The DYNC2H1 knockout in this background creates a tool to dissect the contribution of Hedgehog signaling to ccRCC phenotypes independently of ciliary structural defects, as the genetic ablation directly targets the dynein motor rather than ciliary assembly per se. This model enables studies of how retrograde IFT deficiency alters the balance between Gli activator and repressor forms under conditions of VHL loss, potentially revealing vulnerabilities in tumor cells reliant on paracrine Hh signaling or basal ciliary function.
Applications include investigation of cilia-dependent Hedgehog pathway activation in VHL-deficient renal cancer, screening for ciliary trafficking modulators, and functional dissection of dynein-2 complex assembly. Assays employ immunofluorescence for ciliary markers (ARL13B, acetylated ??-tubulin), Western blotting for DYNC2H1 and Gli proteins, qRT-PCR of Hedgehog targets, and Gli-dependent luciferase reporters. The polyclonal population is suited for pooled functional screens or bulk biochemical analyses where clonal heterogeneity captures editing outcomes. For additional information, contact Ascent Research.
