The DNPEP Knockout HAP1 Polyclonal Cells product provides a CRISPR/Cas9-mediated gene-disrupted polyclonal knockout cell population designed for loss-of-function studies of the DNPEP gene in a haploid human genetic background. This product employs a polyclonal format, representing a heterogeneous pool of edited cells, which enables robust and reproducible investigation of DNPEP-dependent phenotypes without clonal selection artifacts. The knockout model is generated by CRISPR/Cas9-mediated disruption of the DNPEP genomic locus, leading to functional inactivation of the encoded aspartyl aminopeptidase. This population serves as a versatile tool for dissecting DNPEP??s roles in peptide metabolism and immune surveillance pathways.
The host HAP1 cell line is a near-haploid human cell line originally derived from the KBM-7 chronic myeloid leukemia line. Its haploid nature simplifies genetic perturbation because each gene is present in only one copy, eliminating the need for biallelic targeting and reducing compensatory redundancy from a second allele. HAP1 cells grow as an adherent monolayer and are extensively employed in genome-wide screening, functional genomics, and drug target validation due to their streamlined genome and well-characterized proteome. This genetic simplicity makes HAP1 an ideal chassis for generating clean knockout models and for directly linking gene disruption to cellular phenotypes.
DNPEP encodes cytosolic aspartyl aminopeptidase, a zinc-dependent metalloprotease that specifically hydrolyzes N-terminal acidic amino acids (aspartate or glutamate) from peptide substrates. In the renin-angiotensin system, DNPEP functions downstream of renin and ACE, converting angiotensin II into angiotensin III, which then signals through the AT1 receptor to modulate vasoconstriction and electrolyte balance. Notably, DNPEP activity is regulated by zinc cofactor availability, substrate levels such as angiotensin II and enkephalins, and possibly by transcription factors Sp1 and NF-??B, while its protein turnover may involve ubiquitin C and the proteasome complex. Additionally, DNPEP participates in antigen processing by trimming N-terminal residues of endogenous peptides destined for loading onto MHC class I molecules; it interacts with components of the peptide loading complex, including MHC class I heavy chain, TAP1/2, and ??2-microglobulin. Through these activities, DNPEP shapes the MHC class I-bound peptide repertoire and influences immune surveillance.
In the HAP1 haploid context, disruption of DNPEP yields a potent loss-of-function model that unmasks the enzyme??s contributions to both peptidergic signaling and antigen presentation. Abolishing DNPEP function is expected to impair angiotensin II-to- angiotensin III conversion, thereby altering renin-angiotensin pathway outputs, and to perturb enkephalin processing, which may affect opioid peptide signaling. Simultaneously, knockout cells exhibit altered MHC class I surface expression and a modified immunopeptidome, as the supply of properly trimmed epitopes is compromised. This dual impact makes the model particularly valuable for studying cross-talk between hormonal and immune pathways, as well as for exploring how dysregulation of these processes contributes to diseases like hypertension, chronic kidney disease, and evasion of anti-tumor immunity.
Typical applications of the DNPEP Knockout HAP1 Polyclonal Cells include functional genomics to de-orphanize substrates, detailed interrogation of the renin-angiotensin system using angiotensin III ELISA or synthetic substrate cleavage assays, and immunopeptidomic profiling to map alterations in the MHC class I peptide repertoire by mass spectrometry. The polyclonal knockout population is also well-suited for drug target validation, for instance by measuring sensitivity to aminopeptidase inhibitors such as bestatin, and for dissecting the role of aminopeptidases in MHC class I antigen processing via flow cytometric monitoring of MHC class I surface expression. These cells support a wide array of downstream analyses including RT-qPCR and Western blot for knockout confirmation, cell viability and proliferation assays, and mechanistic studies of DNPEP??s links to cardiovascular disease, neurodegeneration, and cancer. For further technical details, ordering information, or custom project inquiries, please contact Ascent Research.
