The DNAAF5 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited heterogeneous cell population with targeted disruption of the DNAAF5 gene, generating a loss-of-function model. Derived from HEK293T cells, this polyclonal pool harbors diverse editing events, enabling functional studies without clonal isolation. These cells are suitable for exploring consequences of DNAAF5 ablation on cytoplasmic preassembly of axonemal dynein arms and ciliary processes.
HEK293T is a human embryonic kidney epithelial cell line transformed with adenovirus 5 DNA and stably expressing SV40 large T antigen. This background provides high transient transfection efficiency and robust protein production, commonly utilized for recombinant protein and viral vector generation. Under serum starvation, HEK293T cells can form primary cilia, making them a useful model for ciliogenesis research. Although not specialized for motile cilia, these cells retain the molecular machinery for dynein arm assembly, enabling investigation of DNAAF5 function in a simplified cellular context.
DNAAF5 encodes a cytoplasmic assembly factor essential for preassembly of axonemal dynein complexes, the motors of motile cilia. It cooperates with other dynein axonemal assembly factors (DNAAF2?C4) and molecular chaperones HSP70/HSP90 to facilitate stepwise folding and assembly of dynein heavy chains (DNAH5, DNAH11) and intermediate chains (DNAI1, DNAI2) into functional dynein arms. Expression of DNAAF5 is regulated by FOXJ1 and RFX transcription factors, key regulators of ciliogenesis. Disruption of DNAAF5 impairs dynein arm formation, leading to defective ciliary motility.
DNAAF5 knockout in HEK293T provides a versatile system to study dynein arm assembly in a non?mucociliary background. Since these cells normally produce non?motile primary cilia, the contribution of DNAAF5 to dynein preassembly can be assessed biochemically and ultrastructurally without complicating beating phenotypes. This model permits reconstitution of wild?type or mutant DNAAF5 for functional rescue and protein interaction studies. The high transfectability of HEK293T also allows overexpression or knockdown of interacting partners to further dissect the preassembly pathway.
This polyclonal knockout pool supports diverse applications: immunofluorescence for ciliary markers (acetylated tubulin, ARL13B); western blotting for dynein subunits; co?immunoprecipitation to map DNAAF5 interactomes; RT?qPCR profiling of ciliogenesis genes; and drug screening to restore dynein assembly. Following ciliogenesis induction, high?speed video microscopy may detect motility defects. The model advances research into primary ciliary dyskinesia (CILD18), Kartagener syndrome, respiratory disease, and infertility. For further details, contact Ascent Research.
