This CRISPR/Cas9-edited polyclonal knockout cell pool targets the GRAMD1A gene in the A-549 human lung adenocarcinoma cell line. The product provides a heterogeneous population of cells with GRAMD1A disruption, enabling loss-of-function studies of this cholesterol-sensing transporter at ER-plasma membrane (ER-PM) contact sites. As polyclonal knockout cells, this population recapitulates the genetic variability inherent to the CRISPR editing process, offering a robust model to study GRAMD1A-dependent phenotypes without clonal selection bias. The knockout model is designed for investigating cholesterol homeostasis and signaling networks critical in lung cancer biology.
A-549 is a widely used adherent epithelial cell line derived from a lung carcinoma of a 58-year-old Caucasian male. These cells display a pseudodiploid karyotype and serve as an established model for human lung adenocarcinoma research, including drug metabolism and cancer signaling studies. The A-549 line??s robust growth characteristics and well-characterized transcriptome make it suitable for genetic perturbation experiments, such as CRISPR/Cas9-mediated knockout, allowing dissection of molecular pathways underlying non-small cell lung cancer. Its epithelial origin provides a relevant context for examining cholesterol transport and membrane contact site dynamics in lung cancer pathophysiology.
GRAMD1A is a cholesterol transport protein localized at ER-plasma membrane contact sites, where it senses cholesterol levels and facilitates non-vesicular lipid transfer. Its activity is regulated by cholesterol availability, SREBP2, Liver X receptor (LXR), and Akt kinase. Upon cholesterol binding and Akt-mediated phosphorylation, GRAMD1A interacts with VAPA/VAPB and OSBP to mediate sterol exchange between organelles. This process directly modulates Akt/mTORC1 signaling and membrane cholesterol distribution, thereby impacting downstream effectors such as YAP/TAZ in the Hippo pathway. Disruption of GRAMD1A uncouples cholesterol sensing from downstream signaling, leading to impaired Akt/mTORC1 activity and altered cellular responses. Additionally, GRAMD1A interacts with STIM1 and ORP1L, linking it to a broader network of ER-PM junctional complexes coordinating lipid and calcium signaling.
In the context of A-549 lung adenocarcinoma cells, GRAMD1A knockout disrupts the finely tuned cholesterol homeostasis required for malignant phenotypes, including proliferation, migration, and survival. Loss of GRAMD1A impairs non-vesicular cholesterol transport, leading to altered membrane lipid composition and attenuation of Akt/mTORC1 pro-growth signals. This perturbation provides a powerful tool to dissect the contribution of ER-PM contact sites to lung cancer progression and to evaluate cholesterol-dependent vulnerabilities in tumor cells. The polyclonal nature of the knockout population further enables the study of cell-to-cell variability in cholesterol handling and signaling adaptation, which is particularly relevant in heterogeneous cancers.
These polyclonal knockout cells are suitable for cholesterol uptake/efflux assays, RNA-seq transcriptomic profiling, and Western blotting for Akt/mTORC1 pathway activity. Immunofluorescence staining of GRAMD1A and other contact site markers, confocal microscopy to visualize ER-PM junction morphology, and flow cytometry for membrane cholesterol levels can be performed. Functional assays such as cell migration and invasion tests directly assess the role of GRAMD1A in metastatic behavior. The knockout model is also ideal for drug testing of cholesterol pathway modulators, including statins and LXR agonists, in a lung cancer background. For detailed product information, technical inquiries, or custom gene-editing services, please contact Ascent Research.
