The ATP8B1 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the ATP8B1 gene. This heterogeneous pool of HEK293T cells enables loss-of-function studies of the encoded P4-ATPase phospholipid flippase, essential for maintaining plasma membrane lipid asymmetry. The polyclonal format avoids clonal artifacts and provides a robust model for investigating ATP8B1 biology and related signaling pathways without the need for single-cell isolation.
HEK293T cells are a human embryonic kidney cell line expressing the SV40 large T antigen, which facilitates high-level protein expression and episomal replication. Originating from renal proximal tubule epithelium, these cells are widely employed for transient transfection, receptor reconstitution, and mechanistic signaling studies. Although non-hepatic, their tractable genetic manipulation makes them a suitable platform for expressing bile acid transporters and nuclear receptors to model hepatobiliary processes.
ATP8B1 encodes an aminophospholipid flippase that translocates phosphatidylserine and phosphatidylethanolamine from the outer to the inner plasma membrane leaflet, a process requiring the accessory subunit CDC50A (TMEM30A). Transcription of ATP8B1 is regulated by bile acid-activated FXR, along with HNF4?? and RXR. Downstream, flippase activity supports proper localization and function of canalicular transporters ABCB11 (BSEP) and ABCB4 (MDR3), and influences FXR-mediated signaling through FGF19 and CYP7A1, thereby controlling bile acid secretion and membrane lipid composition.
In the HEK293T context, ATP8B1 knockout disrupts phosphatidylserine internalization, which can be quantified via Annexin V staining and fluorescent lipid uptake assays. This model permits dissection of ATP8B1?CCDC50A interactions and the impact on membrane asymmetry without liver-specific confounders. It serves as a reductionist system to study the cellular consequences of ATP8B1 loss that underlie cholestatic disorders such as PFIC1 and BRIC1.
Applications include functional phospholipid transport assays using NBD-labeled lipids, drug screening for compounds that restore flippase activity or FXR signaling, and expression analysis of BSEP, MDR3, and FGF19 by RT-qPCR and Western blotting. Knockout verification is performed by Sanger sequencing. These polyclonal cells are ideal for high-throughput screening and pathway interrogation. For additional information, please contact Ascent Research.
