The KLHDC3 Knockout HAP1 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal knockout population derived from the HAP1 cell line, designed for loss-of-function studies of the KLHDC3 gene. This heterogeneous pool of cells, generated through CRISPR/Cas9-mediated gene disruption, enables functional analysis of KLHDC3 without clonal selection, accurately representing population-level biological variability.
HAP1 cells are a near-haploid human male cell line originating from the chronic myeloid leukemia (CML) line KBM-7, which harbors the BCR-ABL fusion oncogene. The cells are haploid for most chromosomes, with disomy only of chromosome 8, making them an ideal platform for haploid genetic screens and gene-trap mutagenesis. The leukemic background provides a relevant context for investigating cancer-driving pathways, and the rapid proliferation of HAP1 cells facilitates high-throughput experimental workflows.
KLHDC3 acts as a substrate-specific adaptor for the CUL3-RBX1 E3 ubiquitin ligase, targeting proteins such as MAP1LC3B (LC3B) and SEPT9 for ubiquitination and proteasomal degradation. This degradation process is tightly controlled by upstream transcriptional regulators SP1 and MYC, which respond to cellular stress signals. Through selective ubiquitination, KLHDC3 governs protein turnover within the ubiquitin-proteasome system and autophagy, impacting cellular homeostasis and stress adaptation.
In HAP1 cells, KLHDC3 knockout disrupts the CUL3-RBX1 ligase??s ability to degrade key substrates, leading to elevated levels of LC3B and potential dysregulation of autophagic flux. Given the BCR-ABL?Cpositive milieu, this model is particularly suited to dissect the intersection of oncogenic signaling, protein quality control, and drug resistance mechanisms in CML. The polyclonal nature preserves cellular diversity, enabling the study of compensatory adaptations to KLHDC3 loss.
This knockout cell population supports diverse experimental applications, including Western blotting for target protein expression, cycloheximide chase assays for degradation kinetics, and ubiquitination assays for E3 ligase activity. Autophagy flux analyses, immunofluorescence-based LC3B puncta quantification, and mass spectrometry?Cbased proteomics can delineate mechanistic consequences of KLHDC3 disruption. The model is also compatible with chemical and genetic screens to identify modifiers of KLHDC3-dependent phenotypes, facilitating drug discovery targeting the ubiquitin-proteasome system. For further information, please contact Ascent Research.
