The AOAH Knockout A-549 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human A-549 lung adenocarcinoma epithelial cell line, designed to disrupt the AOAH gene. This polyclonal pool provides a heterogeneous population with targeted gene disruption, suitable for loss-of-function studies without clonal isolation. The product is intended for investigators examining acyloxyacyl hydrolase (AOAH)-mediated lipopolysaccharide (LPS) detoxification and its role in innate immunity and inflammation.
The A-549 cell line, originally established from a human lung adenocarcinoma, is a widely employed model for respiratory epithelium and lung cancer. These epithelial cells retain type II pneumocyte features, including TLR expression and cytokine secretory capacity, and are routinely used in pulmonary infection, inflammation, and barrier function studies. This host provides a relevant context for studying AOAH function in airway inflammatory disorders such as acute lung injury and asthma.
The AOAH gene encodes acyloxyacyl hydrolase, an enzyme that selectively removes secondary acyl chains from LPS, generating a detoxified form that fails to activate TLR4. Consequently, AOAH functions as a critical negative regulator of TLR4-mediated innate immune signaling. Under LPS stimulation, TLR4 associates with MyD88 and IRAK4, triggering TRAF6-dependent activation of NF-??B and MAP kinases, which drive transcription of pro-inflammatory cytokines including TNF-?? and IL-6. AOAH interacts with LPS in concert with LPS-binding protein and CD14, dampening this cascade and mitigating endotoxic shock. Disruption of AOAH thus removes this brake, yielding cells with heightened LPS responsiveness.
In A-549 cells, AOAH knockout provides a sensitized model for LPS-induced pulmonary inflammation and injury. Lung epithelial cells express functional TLR4, and augmented signaling upon AOAH loss mimics scenarios of uncontrolled inflammation observed in endotoxemia, sepsis, and inflammatory bowel disease. This model is also pertinent to studying asthma exacerbations triggered by bacterial pathogens, where epithelial innate responses play a central role. Additionally, the knockout facilitates dissection of epithelial-intrinsic pathways versus immune cell contributions in acute lung injury models.
Typical applications include LPS-stimulated cytokine ELISA to quantify TNF-?? and IL-6 secretion, NF-??B luciferase reporter assays to gauge transcriptional activation, and western blotting for phospho-p65 or phospho-p38 MAPK to assess signaling intensity. The cells can be analyzed by flow cytometry for surface activation markers or used in endotoxin activity assays to directly measure LPS deacylation capacity. Moreover, the polyclonal knockout population serves as a robust platform for high-throughput screening of anti-inflammatory compounds targeting the TLR4?CNF-??B axis. For additional product details, please contact Ascent Research.
