GRAMD1C Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population designed for loss-of-function studies of GRAMD1C in colorectal adenocarcinoma cells. The product comprises HT29 cells with targeted GRAMD1C gene disruption, enabling robust investigation of cholesterol homeostasis without the need for single-cell cloning.
The HT29 parental cell line originates from a primary colorectal adenocarcinoma of a 44-year-old female and is an established model of the human intestinal epithelium. These adherent cells are characterized by a hypertriploid karyotype, constitutive mucin production, and the ability to undergo enterocytic differentiation when confluent or treated with inducers such as sodium butyrate. This dual capacity for proliferation and differentiation makes HT29 cells ideal for studying cholesterol absorption and metabolism in a physiologically relevant setting.
GRAMD1C is an ER-resident protein that functions as a cholesterol sensor and non-vesicular transporter at ER?Cplasma membrane contact sites. When plasma membrane cholesterol levels rise, the GRAM domain binds sterol, inducing a conformational change that facilitates cholesterol transfer to the ER in concert with the adaptor proteins VAPA and VAPB. Restoring ER cholesterol suppresses SREBP2 activation, thereby downregulating HMGCR and LDLR genes. Additionally, GRAMD1C modulates mTORC1 signaling, integrating lipid availability with cellular growth control. Downstream effects include ACAT-catalyzed cholesterol esterification and lipid droplet biogenesis, which are co-regulated by oxysterol-LXR pathways.
In the HT29 colorectal cancer model, loss of GRAMD1C disrupts the normal sensor-effector loop that couples exogenous cholesterol uptake to intracellular cholesterol homeostasis. This perturbation likely deranges cholesterol esterification, lipid droplet dynamics, and SREBP2-mediated transcription, while concurrently altering mTORC1 activity that controls cell growth and proliferation. As intestinal cells are exposed to varying dietary cholesterol loads, GRAMD1C knockout in HT29 cells offers a powerful system to dissect intestinal cholesterol handling and its links to cancer metabolism.
Researchers can utilize these polyclonal knockout cells in BODIPY-cholesterol trafficking assays, cholesterol esterification quantification, and immunoblotting for SREBP2 and phospho-S6K (mTORC1 marker). Oil Red O staining visualizes neutral lipids, and proximity ligation assays verify ER-PM contact sites. RT-qPCR analysis of HMGCR and LDLR further elucidates transcriptional feedback. These tools position the model for drug screening in atherosclerosis, Alzheimer??s disease, non-alcoholic fatty liver disease, and colorectal cancer. For additional information, please contact Ascent Research.
