The DNAH5 Knockout HEK293T Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human embryonic kidney HEK293T cell line, featuring targeted disruption of the DNAH5 gene. This product provides a heterogeneous pool of cells carrying diverse loss-of-function mutations in DNAH5, generated through non-homologous end joining following Cas9-mediated double-strand cleavage. The polyclonal format offers a robust experimental system for modeling loss of DNAH5 function without the clonal selection artifacts inherent to single-cell-derived lines. Researchers can utilize these cells to investigate the consequences of DNAH5 ablation in a consistently proliferative and easily transfectable host background, enabling a wide range of live-cell and fixed-cell assays.
The host HEK293T cell line is a widely adopted tool in molecular and cellular biology, originating from human embryonic kidney cells immortalized by the SV40 large T antigen. This antigen facilitates episomal replication of plasmids containing the SV40 origin of replication, rendering HEK293T cells exceptionally amenable to transient and stable transgene expression, as well as lentiviral and retroviral production. Although HEK293T cells do not spontaneously form cilia under standard culture conditions, they can be induced to undergo ciliogenesis upon serum starvation or forced expression of master regulators, thereby providing a tunable platform for studying ciliary biology. The robust growth and straightforward manipulation of this line make it a practical host for interrogating the functions of ciliary genes such as DNAH5.
DNAH5 encodes dynein heavy chain 5, a core subunit of the axonemal outer dynein arms that generate the mechanical force driving ciliary and flagellar motility. Within the ciliary axoneme, DNAH5 assembles into large multiprotein complexes alongside interacting factors such as DNAI1, DNAI2, DNAL1, NME8, and CCDC114, which collectively form and stabilize the outer dynein arm structure. Upstream transcriptional regulators, including FOXJ1, RFX2, and MCIDAS, orchestrate the expression of DNAH5 and other ciliary components during ciliogenesis. Once assembled, outer dynein arms undergo ATP-dependent conformational changes that propel microtubule sliding, thereby controlling ciliary beat frequency and, in multiciliated tissues, driving mucociliary clearance. Disruption of DNAH5 consequently abrogates this force-generating machinery, leading to immotile or dyskinetic cilia.
In the HEK293T context, DNAH5 knockout serves as a cell-based model for primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder often caused by mutations in outer dynein arm components. When ciliogenesis is experimentally induced, the polyclonal knockout cells fail to assemble functional outer dynein arms, recapitulating the molecular hallmark of PCD. This system enables dissection of the hierarchical assembly pathway of dynein arms, identification of critical protein-protein interactions, and screening of genetic or pharmacological modifiers that rescue ciliary motility. Moreover, the non-ciliated baseline state allows controlled initiation of the ciliogenesis program, facilitating time-resolved studies of dynein arm preassembly factors and intraflagellar transport.
These polyclonal knockout cells are suited for a diverse array of research applications. They support immunofluorescence microscopy to track ciliary axoneme formation and localization of markers such as acetylated ??-tubulin and ARL13B, enabling visualization of ciliary structure defects. Western blotting and co-immunoprecipitation experiments can probe the steady-state levels and interactions of outer dynein arm proteins, revealing destabilization of the entire complex upon DNAH5 loss. RT-qPCR arrays profile the expression of ciliary genes controlled by FOXJ1 and other transcription factors, while motility assays with high-speed video microscopy quantify ciliary beat frequency following induction. These approaches collectively advance the understanding of ciliary biology and PCD pathogenesis. For additional information or inquiries, please contact Ascent Research.
