GRAMD1A Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population for loss-of-function studies of GRAMD1A in human colorectal adenocarcinoma. This pool of HT29 cells with targeted gene disruption avoids clonal bias and provides a heterogeneous knockout model for robust functional analyses. The polyclonal format is ideal for interrogating GRAMD1A biology without assuming complete gene inactivation.
HT-29 is a human colorectal adenocarcinoma epithelial cell line widely employed in cancer biology, intestinal barrier research, and drug absorption studies. It offers a physiologically relevant system for exploring cholesterol metabolism, signal transduction, and epithelial homeostasis in a gut-derived context.
GRAMD1A encodes an ER-anchored cholesterol sensor/transporter that mediates non-vesicular cholesterol transfer to the plasma membrane at ER-PM contact sites. Its GRAM domain detects PM cholesterol depletion, and through interactions with VAPA/VAPB, it delivers cholesterol to restore membrane lipid order, thereby promoting AKT recruitment and mTORC1 activation. Upstream regulation involves sterol sensing via SREBP2, PPAR??, LXR, and oxysterols, while downstream effects include altered AKT phosphorylation, SREBP target gene expression, and LDL receptor levels. Core pathway components include PI(3,4,5)P3, AKT1, mTOR, RHEB, TSG101, LDLR, and SREBF2, with GRAMD1A interacting with PI(4,5)P2 and OSBP.
In HT29 cells, GRAMD1A disruption impairs cholesterol trafficking to the PM, potentially disrupting lipid raft-dependent PI3K-AKT-mTOR signaling crucial for colorectal cancer cell growth and survival. This knockout model allows dissection of how aberrant cholesterol metabolism fuels tumorigenic processes in intestinal epithelium and affects sterol-regulated gene expression and barrier function.
Applications include studying ER-PM cholesterol transport, lipid raft signaling, and mTORC1-driven metabolism using western blotting for AKT/S6 phosphorylation, fluorescent cholesterol trafficking assays, immunofluorescence colocalization of GRAMD1A/VAPA, filipin staining, RT-qPCR for SREBP targets, cell viability/proliferation, Transwell migration/invasion, and RNA-seq. This polyclonal knockout cell population is a powerful tool for screening modulators of membrane contact sites and investigating cholesterol-related pathways in colorectal cancer. For further details, contact Ascent Research.
