The GRAMD1B Knockout HAP1 Polyclonal Cells product provides a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of GRAMD1B in the HAP1 human cell line. These pooled knockout cells offer a genetically diverse loss-of-function model suitable for functional genomics and pathway interrogation. The polyclonal format avoids clonal artifacts and enables robust assessment of gene ablation effects in cholesterol metabolism and lipid trafficking pathways.
HAP1 is a near-haploid cell line derived from the KBM-7 chronic myeloid leukemia line, exhibiting a fibroblast-like morphology and male karyotype. Its near-haploid genome simplifies knockout generation and phenotypic analysis, while its hematopoietic origin provides a relevant context for studying cholesterol homeostasis. HAP1 cells are widely employed in CRISPR-based screening and gene perturbation studies, offering a reproducible platform for investigating intracellular lipid dynamics.
GRAMD1B encodes Aster-B, a cholesterol transport protein that operates at ER?Cplasma membrane contact sites. It senses plasma membrane cholesterol via its GRAM domain, triggering interaction with ER-resident proteins VAPA and VAPB. This facilitates non-vesicular cholesterol transfer to the ER, directly modulating SREBP-2 activity. SREBP-2 controls expression of HMGCR, LDLR, and other cholesterol synthesis and uptake genes. GRAMD1B is regulated by SREBP2, 25-hydroxycholesterol, and LXR agonists, and it interacts with OSBP. Thus, it functions as a key regulator of cholesterol homeostasis, connecting membrane cholesterol status to ER-driven transcriptional responses.
In HAP1 cells, GRAMD1B disruption is expected to impede cholesterol delivery from the plasma membrane to the ER, altering ER cholesterol pools and SREBP-2 cleavage. This can dysregulate downstream targets such as HMGCR and LDLR, and affect cholesterol esterification by ACAT. The near-haploid background enhances phenotypic clarity, making HAP1 an ideal host for deciphering the role of non-vesicular cholesterol transport in lipid-sensitive signaling.
Applications include fluorescence-based cholesterol uptake and distribution assays (e.g., filipin staining, fluorescent cholesterol analogs), SREBP-2 cleavage immunoblotting, qPCR for target genes, co-immunoprecipitation of GRAMD1B?CVAP complexes, and lipidomic profiling. This model is relevant for investigations into atherosclerosis, NAFLD, Niemann-Pick type C, and dyslipidemia, and for drug discovery targeting cholesterol transport. For additional details, contact Ascent Research.
