The GRAMD1A Knockout SK-HEP-1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for functional loss-of-function analysis of the GRAMD1A gene. Through CRISPR/Cas9-mediated gene disruption, a heterogeneous pool of edited SK-HEP-1 cells is generated, collectively exhibiting reduced or absent GRAMD1A expression. This polyclonal format avoids the time-intensive steps of single-cell cloning, offering an efficient system for preliminary and high-throughput studies. The product provides a powerful model to dissect the roles of GRAMD1A in cholesterol sensing, non-vesicular transport, and hepatocellular carcinoma physiology.
The SK-HEP-1 host cell line is an epithelial-like immortalized cell line derived from the ascitic fluid of a male patient with liver adenocarcinoma. Originating from a metastatic site, SK-HEP-1 cells are widely used to study aggressive hepatocellular carcinoma phenotypes, drug metabolism, and lipid biology. Their robust growth and well-defined culture conditions make them amenable to genetic manipulation and downstream phenotypic assays. GRAMD1A knockout in this context creates a clinically relevant model to investigate how cholesterol trafficking and ER?CPM contact sites impact liver cancer cell signaling and survival.
GRAMD1A functions as a cholesterol sensor and transporter at ER?CPM contact sites. Its StART domain detects PM cholesterol, triggering binding to ER VAPA/VAPB proteins and enabling non-vesicular cholesterol transfer to the ER. This cholesterol supply regulates SREBP2 cleavage: high ER cholesterol retains SREBP2 as an inactive precursor, while low cholesterol permits cleavage and activation of genes including HMG-CoA reductase and the LDL receptor. Upstream regulators include PM cholesterol levels, phosphatidylinositol (4,5)-bisphosphate, and LXR agonists. Downstream, GRAMD1A influences ACAT-mediated esterification and ER stress responses, and collaborates with lipid transfer proteins OSBP and ORP5/8.
In hepatocellular carcinoma, cholesterol metabolism is frequently dysregulated to support membrane biogenesis and proliferation. GRAMD1A knockout in SK-HEP-1 cells enables dissection of how impaired ER?CPM cholesterol trafficking impacts viability, SREBP2-dependent oncogenic signaling, and lipid raft composition. This system can uncover synthetic lethal interactions arising from defective cholesterol sensing, guiding therapeutic strategies for liver cancers linked to dyslipidemia and metabolic syndrome. Comparative analysis with control cells can reveal compensatory lipid transport pathways and altered sensitivity to lipid-lowering agents.
This GRAMD1A knockout cell pool is suitable for diverse functional assays: cholesterol efflux, filipin staining, SREBP2 cleavage western blot, LC-MS lipidomics, co-IP of VAP-GRAMD1A, and immunofluorescence of ER/PM markers. Phenotypic readouts include MTT viability under lipid stress, flow cytometry for LDL uptake, and RNA-seq of cholesterol pathway genes. The polyclonal format accelerates high-throughput screening for modulators of non-vesicular cholesterol transport or SREBP2 activation. For additional technical information, please contact Ascent Research.
