The ANO6 Knockout HEK293 Cell Line is a CRISPR/Cas9-mediated gene-disrupted cell line derived from the widely used human embryonic kidney HEK293 epithelial cell line. This knockout model enables loss-of-function studies of anoctamin-6 (ANO6), a critical calcium-activated ion channel and phospholipid scramblase, without altering the immortalized adherent growth characteristics of the parental line.
HEK293 cells are an established immortalized line generated by transformation of human embryonic kidney cells with sheared adenovirus type 5 DNA. They exhibit epithelial-like morphology and adherent growth, and they are extensively employed for recombinant protein expression, viral packaging, and calcium signaling studies due to their robust transfection efficiency and well-characterized physiology. This cellular background provides a reliable platform for examining the specific contributions of ANO6 to phospholipid scrambling and coagulation-related processes.
ANO6 encodes a dual-function protein that acts as a calcium-activated non-selective cation channel and a phospholipid scramblase. Upon binding of elevated intracellular calcium, which can be mobilized through PLCgamma-mediated IP3 production and STIM1/ORAI1 store-operated entry, ANO6 undergoes a conformational shift that facilitates bidirectional phospholipid translocation, culminating in phosphatidylserine (PS) exposure on the outer membrane leaflet. This externalized PS serves as a docking site for the prothrombinase complex components Factor Va and Factor Xa, promoting thrombin generation. ANO6 function is regulated by upstream signals such as thrombin, collagen, P2Y receptors, and Podoplanin/RhoA/ROCK kinase cascades, and it cooperates with interacting partners including integrin beta-3, flotillin-2, P2X7 receptor, and the Xkr8 scramblase. Downstream, PS externalization enables lactadherin and annexin V binding, microparticle shedding, and cell-cell fusion, linking ANO6 to platelet activation, apoptotic clearance, and blood coagulation.
Employing HEK293 cells as the host for ANO6 disruption leverages their tractable calcium signaling machinery and high transfection efficiency. While HEK293 cells do not fully recapitulate platelet-specific pathways, they express many core components of calcium-regulated phospholipid scrambling, making this knockout line an ideal platform for reconstituting and dissecting ANO6-dependent functions. Loss of ANO6 in this background abrogates calcium-induced phosphatidylserine externalization and microparticle generation, allowing researchers to attribute such phenomena directly to anoctamin-6 activity. This model also facilitates overexpression of interacting proteins or disease-associated mutants to probe structure?Cfunction relationships and scramblase regulation.
Researchers can apply this ANO6 knockout HEK293 line to investigate the molecular underpinnings of Scott syndrome by assessing phosphatidylserine exposure via annexin V flow cytometry and microparticle release assays. The model is also amenable to calcium imaging with Fluo-4 AM, patch-clamp electrophysiology to characterize channel properties, and prothrombinase generation assays to evaluate coagulation function. Furthermore, it supports studies on viral entry that exploits scramblase-mediated membrane changes, cancer anoikis resistance mechanisms, and high-throughput screening for scramblase modulators. CRISPR editing is confirmed by Sanger sequencing and immunoblotting, ensuring reliable gene disruption. For further details or inquiries, please contact Ascent Research.
