The KLHL25 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population targeting the KLHL25 gene in the HAP1 human near-haploid cell line. This heterogeneous pool of cells harbors diverse indel mutations at the target locus, generated by non-homologous end joining, avoiding clonal selection biases. As a polyclonal knockout, it enables functional studies of KLHL25 loss-of-function in ubiquitin-mediated proteolysis, hypoxia signaling, and actin dynamics. The cells serve as a versatile reagent for mechanistic assays and phenotypic screens.
HAP1 cells are near-haploid fibroblastoid cells derived from the KBM-7 chronic myelogenous leukemia cell line, with a haploid karyotype for most chromosomes except a disomic fragment of chromosome 15. Originating from a male donor, these adherent cells simplify loss-of-function studies because gene disruption typically yields complete functional knockout without allelic compensation. Their stable growth and suitability for transfection and high-throughput imaging make them a preferred model for genetic perturbation screens. In this haploid background, KLHL25 knockout provides a defined system for investigating gene function.
KLHL25 encodes a BTB-Kelch adaptor protein that recruits HIF-1?? to the CUL3-RBX1 RING E3 ubiquitin ligase complex, promoting its ubiquitination and proteasomal degradation in an oxygen-independent manner. This activity complements the VHL pathway and controls HIF-1?? transcriptional output. The process involves NEDD8 modification of CUL3, and E1/E2 enzymes such as UBA1 and UBE2M. KLHL25 also interacts with actin cytoskeletal components, linking ubiquitin signaling to cytoskeletal regulation. Upstream regulators include the CUL3 complex, NEDD8 conjugation machinery, and hypoxia, while downstream targets encompass HIF-1?? protein stability and HIF-1 target gene expression.
The haploid nature of HAP1 cells ensures that KLHL25 disruption results in near-complete loss of function, enabling robust analysis of HIF-1?? regulation without confounding heterozygous effects. This model is particularly valuable for studying the interplay between oxygen-independent and PHD-VHL-dependent HIF-1?? degradation pathways. Given the leukemia origin of HAP1 cells, the knockout is relevant for cancer research where HIF-1?? drives tumor progression. The polyclonal population also supports genetic screens for synthetic lethality and drug sensitization in a clean genetic background.
These cells enable applications such as HIF-1?? Western blotting under hypoxia or proteasome inhibition, RT-qPCR of HIF-1 target genes, and HRE-reporter assays. Co-immunoprecipitation can probe KLHL25-CUL3-HIF-1?? interactions, and ubiquitination assays measure E3 ligase activity. Actin cytoskeleton studies, cell proliferation, apoptosis, and migration assays under normoxic/hypoxic conditions are also facilitated. The polyclonal format is ideal for modifier screens and drug response studies in leukemia models. For further information, please contact Ascent Research.
