The DPH6 Knockout HEK293T Polyclonal Cells product is a CRISPR/Cas9-edited polyclonal knockout population derived from the human embryonic kidney HEK293T cell line. This model features targeted disruption of the DPH6 gene, which encodes an essential enzyme for diphthamide biosynthesis. The polyclonal format provides a mixed population of edited cells, enabling robust analysis of gene function without clonal selection biases. The knockout is generated using CRISPR/Cas9-mediated gene disruption, resulting in a loss-of-function model suitable for studying the role of DPH6 in post-translational modification of eukaryotic elongation factor 2 (eEF2).
The HEK293T host cell line, derived from HEK293 cells stably expressing the SV40 large T antigen, is a widely utilized human embryonic kidney derivative. This feature enables episomal replication of plasmids containing the SV40 origin, leading to high-level transient protein expression and efficient production of lentiviral and retroviral vectors. HEK293T cells are extensively employed in signal transduction, protein expression, and virology research due to their rapid growth, high transfection efficiency, and well-characterized cellular biochemistry. The DPH6 knockout introduced into this background preserves these advantageous traits while allowing dissection of diphthamide-dependent processes.
DPH6 catalyzes the final step in the diphthamide biosynthesis pathway: the ATP-dependent amidation of diphthine to diphthamide on histidine-715 of eEF2. This modification is essential for the fidelity of translation elongation and renders cells sensitive to ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas exotoxin A. DPH6 acts downstream of the DPH1?CDPH5 enzyme complex that generates diphthine from a histidine precursor, using ammonia as the nitrogen donor. The resulting diphthamide modification on eEF2 is the target of toxin-mediated ADP-ribosylation, which inactivates eEF2 and halts protein synthesis. Thus, DPH6 is a critical node connecting diphthamide biosynthesis to translational control and toxin susceptibility.
In the HEK293T context, loss of DPH6 ablates diphthamide formation, yielding eEF2 that lacks the modification. This knockout model enables investigation of how diphthamide deficiency influences translation elongation dynamics, ribosomal function, and cellular responses to toxin exposure. Because HEK293T cells are of human kidney origin, they provide a physiologically relevant platform for studying the consequences of defective diphthamide synthesis, which has been linked to developmental disorders and susceptibility to bacterial toxins. Moreover, the ability to perform high-efficiency transfection and viral packaging in this line facilitates reconstitution experiments and toxin-based selection strategies.
Researchers can employ this DPH6 knockout polyclonal population in various assays to probe diphthamide biology. Western blotting with anti-diphthamide antibodies confirms loss of the modification, while ADP-ribosylation assays using diphtheria toxin or Pseudomonas exotoxin A demonstrate functional resistance. Polysome profiling and puromycin incorporation assays assess effects on translation elongation rates and ribosomal occupancy. Cell viability assays in the presence of toxins provide a direct readout of DPH6 activity. These cells are well-suited for studying translational fidelity, ribosome quality control, and the role of diphthamide in cellular physiology. For further details or to discuss custom applications, please contact Ascent Research.
