The ATP8A1 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human colorectal adenocarcinoma cell line HT29. This loss-of-function model is designed to disrupt ATP8A1 expression, enabling studies of aminophospholipid flippase activity in a well-characterized intestinal epithelial context. The polyclonal nature of the knockout pool captures a range of editing events, providing a robust system for functional genomics without clonal bias.
HT29 cells, originally isolated from a 44-year-old female colorectal adenocarcinoma, exhibit epithelial morphology and are widely employed as an in vitro model for intestinal epithelial biology and colorectal cancer research. These cells can differentiate into enterocyte-like cells upon reaching confluence or following butyrate treatment, and they produce mucin, making them particularly suitable for investigating differentiation-dependent processes, barrier function, and drug transport mechanisms.
ATP8A1 is a P4-ATPase flippase that forms a heterodimer with TMEM30A or TMEM30B to transport phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the plasma membrane, upholding lipid asymmetry. Its activity is regulated by EGF, protein kinase C, and its beta subunits. ATP8A1 is essential for EGFR endocytosis and routing??it promotes internalization and recycling of the receptor, thereby maintaining MAPK/ERK and AKT signaling. In the absence of ATP8A1, EGFR trafficking is disrupted, leading to reduced MAPK1/3 and AKT1 phosphorylation, impaired cell migration, and increased phosphatidylserine exposure. The flippase also interacts with clathrin adaptor AP-2, indicating a role in clathrin-mediated endocytosis.
In HT29 cells, which harbor mutant APC and are sensitive to EGFR-mediated proliferation, knockout of ATP8A1 provides a physiologically relevant platform to dissect the interplay between phospholipid transport and oncogenic signaling in colorectal cancer. The model is particularly valuable for studying how lipid asymmetry impacts EGFR trafficking and downstream oncogenic pathways, and for assessing the contribution of flippase activity to cancer cell migration, invasion, and metastasis. Additionally, the HT29 differentiation capacity allows for examining ATP8A1 function in polarized epithelia.
This knockout model supports applications such as western blotting for EGFR, p-ERK, and p-AKT; annexin V flow cytometry for phosphatidylserine externalization; immunofluorescence tracking of EGF uptake; transwell migration and invasion assays; and chemical screening for flippase inhibitors. Additionally, the cells enable co-immunoprecipitation of ATP8A1-TMEM30A complexes, RT-qPCR of downstream targets, RNA-sequencing, and viability assays with chemotherapeutics. For more details, contact Ascent Research.
