GRAMD1B Knockout HEK293T Polyclonal Cells provide a genetically engineered human cell population generated by CRISPR/Cas9-mediated disruption of the GRAMD1B gene. This polyclonal knockout model comprises a heterogeneous mixture of edited cells, each harboring targeted gene disruption events that collectively abolish GRAMD1B expression. The product is supplied as a ready-to-use polyclonal cell stock, enabling researchers to investigate the loss-of-function consequences of GRAMD1B deficiency in a well-characterized cellular background without the need for clonal isolation.
The host cell line, HEK293T, is a derivative of human embryonic kidney 293 cells that constitutively expresses the SV40 large T antigen. This immortalized epithelial line is valued for its high transfectability, rapid growth, and robust capacity for heterologous protein production and lentiviral packaging. These features make it an ideal platform for transient transfection, stable expression, and functional reconstitution experiments, facilitating detailed structure-function analyses of proteins such as GRAMD1B.
GRAMD1B (also designated Aster-B) functions as a non-vesicular cholesterol transporter at endoplasmic reticulum?Cplasma membrane contact sites. Its GRAM domain senses increased plasma membrane cholesterol, triggering a conformational change that enables interaction with ER-resident VAPA and VAPB proteins and docking at the plasma membrane. This tethering facilitates the rapid transfer of cholesterol to the ER, where it serves as a substrate for ACAT1-mediated esterification and suppresses SREBP cleavage, thereby downregulating cholesterol biosynthesis and uptake. Consequently, GRAMD1B acts downstream of cellular cholesterol depletion and insulin signaling, and its transport activity directly influences the activation of LXR and the expression of SREBP target genes, including the LDL receptor.
In the HEK293T context, disruption of GRAMD1B is expected to impair cholesterol sensing at the plasma membrane and attenuate ER cholesterol flux, leading to compensatory SREBP activation and altered lipid metabolism. This model provides a simplified, transcriptionally active system to dissect the molecular determinants of GRAMD1B-mediated sterol transport and to explore its crosstalk with insulin signaling and other pathways. Because HEK293T cells lack tissue-specific constraints, this knockout population offers a clean background for investigating fundamental mechanisms of non-vesicular lipid transfer and for overexpression-based rescue experiments.
Key applications include cholesterol trafficking assays using fluorophore-tagged cholesterol (e.g., DHE or BODIPY-cholesterol), filipin staining for cellular free cholesterol localization, and quantification of cholesterol via Amplex Red. The model enables assessment of SREBP processing by immunoblot, ACAT activity assays, and luciferase reporter studies for SREBP transcriptional activity. Co-immunoprecipitation with VAPA/VAPB, immunofluorescence, and proximity ligation can probe membrane contact site architecture. Moreover, the cells are suitable for LDL uptake assays by flow cytometry and for RT-qPCR profiling of SREBP target genes. For further details, please contact Ascent Research.
