The GRAMD1C Knockout SK-HEP-1 Polyclonal Cells comprise a heterogeneous population of SK-HEP-1 cells engineered via CRISPR/Cas9-mediated gene disruption to introduce loss-of-function mutations in the GRAMD1C locus. This polyclonal knockout pool enables the study of GRAMD1C-dependent functions without the selective pressure of clonal isolation, preserving the inherent genetic diversity of the parental line. As a gene-edited polyclonal cell population, this product is suitable for pooled knockout experiments and population-level phenotypic analyses.
The parental SK-HEP-1 cell line is a human hepatic adenocarcinoma epithelial cell line originally isolated from the ascites of a 52-year-old Caucasian male. These cells are widely utilized as an in vitro model for hepatocellular carcinoma (HCC), exhibiting tumorigenic properties and retaining key metabolic features of liver cancer cells. Their epithelial origin and robust growth characteristics make them amenable to functional studies of cholesterol metabolism and signal transduction in a cancer-relevant context.
The GRAMD1C gene encodes Aster-C, a sterol transfer protein critical for non-vesicular cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER). GRAMD1C is recruited to membrane contact sites through its GRAM domain, which senses phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and mediates cholesterol extraction and delivery via interaction with VAMP-associated proteins VAPA and VAPB. Upon arrival at the ER, cholesterol levels govern the processing of sterol regulatory element-binding protein 2 (SREBP2), a master transcription factor. Under conditions of adequate ER cholesterol, SREBP2 is retained in the ER by SCAP?CInsig complexes; cholesterol depletion triggers SREBP2 cleavage and nuclear translocation, leading to transcriptional upregulation of genes including HMGCR (HMG-CoA reductase) and LDLR (low-density lipoprotein receptor). Thus, GRAMD1C functions as a key upstream regulator of cellular cholesterol homeostasis, integrating plasma membrane lipid cues with ER-resident feedback machinery.
In the context of SK-HEP-1 hepatocellular carcinoma cells, disruption of GRAMD1C provides a valuable tool to dissect the role of cholesterol trafficking in liver cancer biology. Hepatocellular carcinomas frequently exhibit altered lipid metabolism, and aberrant cholesterol flux may contribute to tumor proliferation, survival, and drug resistance. By eliminating GRAMD1C-mediated cholesterol transport, researchers can interrogate whether HCC cells rely on non-vesicular lipid transfer pathways to sustain SREBP2 activity and lipogenic programs. This model enables investigation into potential metabolic vulnerabilities or adaptive mechanisms that cancer cells employ to maintain cholesterol homeostasis, offering insights into new therapeutic targets.
This polyclonal knockout cell population accommodates diverse experimental approaches, including filipin staining for free cholesterol visualization, cholesterol uptake/efflux assays, and western blot analysis of SREBP2 processing and SCAP expression. Transcriptional outputs of SREBP2 activity, such as HMGCR and LDLR, can be measured by RT-qPCR. Protein?Cprotein interactions at ER?Cplasma membrane contact sites are assessable via co-immunoprecipitation of VAP proteins, complemented by immunofluorescence. Lipidomic profiling, PI(4,5)P2 binding assays, and oil red O staining further enable comprehensive dissection of lipid phenotypes. For further technical information and order inquiries, please contact Ascent Research.
