The EBP Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for loss-of-function studies of the EBP gene in a human near-haploid background. This product comprises a heterogeneous pool of HAP1 cells harboring targeted disruption of the EBP locus, providing a flexible and robust tool for interrogating sterol biosynthesis without clonal selection. The polyclonal format captures diverse editing outcomes, enabling the study of gene knockout effects at the population level and reducing clonal artifacts. By eliminating EBP function, researchers can directly assess the consequences of disrupted cholesterol biosynthesis in a physiologically relevant human cellular context.
The HAP1 cell line is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia (CML) line, offering a simplified genetic background ideal for knockout studies. Its haploid nature reduces genetic redundancy and facilitates unambiguous genotype-phenotype correlations. HAP1 cells retain key signaling and metabolic pathways, making them a versatile model for cancer biology and metabolic research. The CML origin provides a platform to study gene function in the context of leukemogenesis, while the near-haploidy ensures that gene disruptions often lead to complete loss of function without the complexity of diploid compensation. This background is particularly suited for sterol biosynthesis investigations, as HAP1 cells maintain active cholesterol metabolism.
EBP encodes sterol ??8-??7 isomerase, an enzyme catalyzing the conversion of ??8-sterols to ??7-sterols in the post-squalene segment of cholesterol biosynthesis. This reaction occurs immediately downstream of SC5D (lathosterol oxidase) and upstream of DHCR7 (7-dehydrocholesterol reductase) in the linear pathway leading to cholesterol. EBP activity is controlled by the SREBF2 transcription factor, which is regulated by the INSIG1-SCAP-SREBP pathway in response to cellular sterol levels and liver X receptor signaling. Upon cholesterol depletion, SREBF2 is cleaved and translocates to the nucleus, where it activates transcription of EBP and other cholesterogenic genes, including HMGCR, SQLE, and CYP51A1. The isomerase product, lathosterol, serves as the immediate substrate for SC5D, and subsequent steps produce 7-dehydrocholesterol, which is reduced by DHCR7 to yield cholesterol. Thus, EBP operates within a tightly regulated metabolic network, interacting with NSDHL, SC5D, and DHCR7 to maintain sterol homeostasis. Disruption of EBP leads to accumulation of 8-dehydrocholesterol and depletion of cholesterol, impacting membrane integrity and precursor supply for steroid hormones and oxysterols.
In the HAP1 leukemia cell model, EBP knockout has profound implications for cholesterol metabolism and cell physiology. Chronic myeloid leukemia cells are particularly dependent on cholesterol for proliferation, and perturbation of the cholesterol biosynthesis pathway may influence leukemogenic signaling. HAP1 cells with EBP disruption can model the metabolic defects observed in X-linked dominant chondrodysplasia punctata (CDPX2), a disorder characterized by sterol imbalance. The near-haploid background allows direct observation of loss-of-function phenotypes without the confounding effects of a second allele, making it a powerful system to link EBP deficiency to accumulation of 8-dehydrocholesterol and the subsequent cascade of signaling alterations. This model facilitates the investigation of cholesterol-dependent processes such as membrane raft formation, vesicular trafficking, and protein prenylation, all of which are critical in leukemia cell survival and proliferation. The knockout cells also provide a platform to study the regulatory feedback mechanisms involving SREBF2 and INSIG1-SCAP, shedding light on how sterol sensing adapts in the absence of a key enzyme.
Researchers can employ this knockout model for a wide range of applications, including detailed sterol profiling by LC-MS to quantify intermediates such as lathosterol and 8-dehydrocholesterol, and for assessing cholesterol distribution by filipin staining. The cells are suitable for drug screening campaigns targeting sterol isomerase or downstream enzymes, and for functional rescue experiments by reintroducing wild-type or mutant EBP. Gene expression analysis by RT-qPCR and validation of protein-level changes via western blotting allow examination of SREBF2 target genes and pathway compensation. Under lipid-depleted conditions, MTT assays can be used to evaluate cell viability and proliferation dependency on cholesterol synthesis. Moreover, the polyclonal pool is useful for studying population-level responses to pharmacological inhibition of the pathway, providing insights into sterol regulatory networks in cancer metabolism. For further information or to discuss custom applications, please contact Ascent Research.
