Pik3c3 Knockout 3T3-L1 Cell Line

The Pik3c3 Knockout 3T3-L1 Cell Line is a CRISPR/Cas9-engineered mouse cell model in which the Pik3c3 gene has been disrupted to abolish functional expression of the class III phosphatidylinositol 3-kinase catalytic subunit VPS34. This stable gene-edited in vitro system is generated in 3T3-L1 cells, a fibroblast-like preadipocyte cell line, and is designed for mechanistic studies of autophagy, endomembrane trafficking, lysosomal biology, and metabolic regulation in a well-established adipocyte precursor background.

3T3-L1 cells are derived from murine embryonic fibroblast-like precursors and are widely used because they undergo hormonally induced differentiation into adipocyte-like cells with robust lipid accumulation and insulin-responsive glucose metabolism. As a result, this host line is a standard experimental platform for investigating adipogenesis, lipid droplet formation, adipokine-related biology, nutrient sensing, and cellular responses relevant to obesity, insulin resistance, type 2 diabetes, and fatty liver disease. The model also provides a tractable system for analyzing autophagy-dependent remodeling events that accompany differentiation and metabolic adaptation.

PIK3C3/VPS34 is the catalytic core of class III PI3K complexes that generate phosphatidylinositol 3-phosphate on nascent autophagic and endosomal membranes. Its activity is regulated by nutrient deprivation, amino acid availability, AMPK, MTOR, and ULK1, and it functions in multiprotein complexes with PIK3R4/VPS15, BECN1, ATG14, UVRAG, RUBICON, NRBF2, and AMBRA1. Through these assemblies, VPS34 acts upstream of WIPI2 recruitment, DFCP1-positive omegasome formation, LC3 lipidation, SQSTM1/p62 turnover, autophagosome formation, endosome maturation, and lysosome-dependent cargo degradation. Because these processes are central to membrane dynamics and stress adaptation, Pik3c3 loss is relevant to metabolic disease, neurodegeneration, cancer, and lysosomal dysfunction research.

In the 3T3-L1 context, Pik3c3 disruption provides a useful model to examine how defective phosphatidylinositol 3-phosphate production alters preadipocyte homeostasis and adipocyte differentiation-associated remodeling. The model can support studies on how autophagosome nucleation and endolysosomal trafficking influence lipid storage, insulin-responsive pathways, and nutrient stress responses in adipocyte-lineage cells. It is also suitable for probing pathway dependence within BECN1-ATG14 or UVRAG-containing VPS34 complexes and for comparing autophagy-linked versus endosomal functions during metabolic adaptation.

Representative applications include western blot analysis of LC3-I/LC3-II conversion and SQSTM1 accumulation, immunofluorescence imaging of LC3, WIPI2, and LAMP1 localization, autophagic flux studies using lysosomal inhibitors, and phosphatidylinositol 3-phosphate reporter imaging. Researchers may also combine this model with Oil Red O staining, RT-qPCR of adipogenic and stress-response genes, RNA-seq, glucose uptake or mitochondrial function assays, electron microscopy of autophagic structures, and co-immunoprecipitation of VPS34 complex components to define Pik3c3-dependent signaling and trafficking mechanisms. Researchers may contact Ascent Research for additional technical information, product details, or related gene-edited cell models.