The AOAH Knockout HAP1 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal knockout cell population targeting the AOAH gene in the near-haploid human HAP1 cell line. This genetically disrupted model provides a powerful loss-of-function tool for investigating the role of acyloxyacyl hydrolase in lipopolysaccharide (LPS) detoxification and innate immune regulation. The polyclonal composition ensures a diverse representation of editing events across the population without selection for single-cell clones, making it suitable for pooled functional screens and robust population-level assays.
The HAP1 parental cell line is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia line, harboring the BCR-ABL translocation. Its male origin and haploid karyotype for most chromosomes make it an exceptional platform for genetic studies, particularly haploid genetic screens and functional genomics. The absence of a second allele in many genes simplifies genotype-phenotype correlations, while the leukemic background provides a relevant context for studying signaling pathways involved in cell proliferation and survival.
AOAH encodes acyloxyacyl hydrolase, which selectively removes secondary fatty acyl chains from the lipid A moiety of bacterial LPS, detoxifying endotoxin. In wild-type cells, LPS binds LBP and CD14, engaging the TLR4/MD2 complex to activate MyD88-dependent NF-??B signaling and transcription of pro-inflammatory cytokines (TNF-??, IL-6, IL-1??). AOAH negatively regulates this pathway: inactivated LPS reduces TLR4 complex activation, downregulates NF-??B, and attenuates cytokine production, contributing to endotoxin tolerance. AOAH expression is itself upregulated by LPS, TLR4, NF-??B, and cytokines like TNF-?? and IL-1??, forming feedback loops, while serum lipoproteins modulate LPS availability.
AOAH knockout in the HAP1 haploid background is particularly valuable because it eliminates the possibility of heterozygosity and allows a clean assessment of AOAH’s non-redundant roles. This model enables researchers to dissect how complete loss of LPS deacylation affects endotoxin-driven signaling without the confounding effects of compensatory alleles. It is ideally suited for genetic screens to identify synthetic lethal interactions or chemical sensitivities that rely on AOAH deficiency, and for studying chronic inflammation or endotoxin hypersensitivity in a simplified genetic context.
Researchers can employ this knockout population in LPS detoxification assays, NF-??B reporter systems, ELISA-based cytokine quantification, Western blotting for phospho-NF-??B p65, flow cytometry for TLR4 expression, and RT-qPCR for cytokine gene transcription. This enables functional dissection of innate immune signaling, screens for inflammatory modulators, and host?Cpathogen interaction studies relevant to Gram-negative bacteria. For further information, contact Ascent Research.
