The DPH7 Knockout HAP1 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HAP1 cell line, engineered to disrupt the DPH7 gene. This loss-of-function model targets DPH7, which encodes a methylesterase essential for the final step of diphthamide biosynthesis. The polyclonal format provides a heterogeneous pool of knockout cells, enabling robust functional studies without clonal selection artifacts. This product is optimized for investigating diphthamide-dependent pathways, translation elongation, and cellular responses to ADP-ribosylating toxins.
The host HAP1 cell line is a near-haploid, adherent cell line derived from the KBM-7 chronic myeloid leukemia (CML) line. These cells are BCR-ABL1 positive, male, and maintain a stable haploid karyotype, making them a powerful tool for functional genomic screens and genetic interaction mapping. The haploid nature facilitates unambiguous gene disruption and enhances the efficiency of CRISPR-based knockout studies, as only a single allele requires targeting to achieve complete loss of function. HAP1 cells retain key signaling networks relevant to CML, providing a disease-relevant context for studying diphthamide biology.
DPH7 functions as a methylesterase that catalyzes the demethylation of diphthine-modified translation elongation factor 2 (EEF2) to yield mature diphthamide. This unique post-translational modification, formed through a highly conserved biosynthetic pathway involving DPH1?CDPH7, is critical for ribosomal translocation fidelity during protein synthesis. DPH7 acts downstream of DPH5 and directly interacts with the diphthine-EEF2 intermediate. Diphthamide serves as the target for ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas exotoxin A, which inactivate EEF2 and halt translation. The pathway integrates methionine salvage via S-adenosylmethionine (SAM) and relies on sequential enzymatic steps, with DPH7 executing the terminal demodification required for functional diphthamide.
Within the HAP1 CML background, DPH7 knockout provides a unique platform to dissect diphthamide-dependent cellular processes. Loss of DPH7 abolishes diphthamide formation on EEF2, conferring resistance to diphtheria toxin while potentially altering global translation dynamics. This model allows researchers to explore how diphthamide status affects protein synthesis fidelity, cell growth, and stress responses in a leukemia-relevant context. The haploid nature of HAP1 cells simplifies the generation of double knockouts for synthetic lethality screens, enabling systematic mapping of genetic interactions with DPH7 and other translation-related factors.
Typical applications include functional dissection of diphthamide biosynthesis using toxin sensitivity assays, co-immunoprecipitation with DPH5, and mass spectrometry-based detection of diphthamide modification. Western blotting for EEF2 and its modified forms can assess pathway integrity, while protein synthesis assays evaluate translational impact. RT-qPCR for DPH7 expression and immunofluorescence for EEF2 localization complement these approaches. The model is also suited for drug target validation and high-throughput genetic screens exploiting the haploid genome. For further technical details, please contact Ascent Research.
