The GRAMD2A Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population engineered from the HEK293T human embryonic kidney line. They carry a loss-of-function disruption of GRAMD2A, a gene encoding a GRAM domain-containing lipid transfer protein. The polyclonal format delivers a heterogeneous knockout pool for efficient functional screening of GRAMD2A-dependent processes without clonal selection. This model is suited for autophagy, lipid trafficking, and membrane contact site investigations.
HEK293T cells derive from human embryonic kidney cells transformed with adenovirus type 5 DNA and constitutively express the SV40 large T-antigen, enabling episomal replication of plasmids bearing the SV40 origin. This feature supports robust transient protein expression and viral packaging, establishing HEK293T as a workhorse for molecular and cellular studies. The cells exhibit fast growth and high transfection efficiency, providing a reliable platform for GRAMD2A loss-of-function analysis.
GRAMD2A facilitates non-vesicular lipid exchange at endoplasmic reticulum?Corganelle membrane contact sites, with roles in autophagosome biogenesis and cholesterol homeostasis. The gene is transcriptionally regulated by TFEB, SREBP1, and PPAR?? and functions downstream of the mTORC1?CAMPK?CULK1 axis. GRAMD2A interacts with autophagy initiation factors ATG16L1 and BECN1, and cholesterol-handling proteins NPC1 and STARD3. It promotes expression of LC3B, SQSTM1/p62, PLIN2, and ABCA1. Its disruption is expected to compromise autophagic flux and distort intracellular cholesterol distribution.
In HEK293T cells, GRAMD2A knockout offers a relevant context to study the interplay between lipid signals and autophagy. The line??s well-characterized autophagy machinery and active cholesterol synthesis pathways facilitate direct measurement of changes in LC3B lipidation, SQSTM1/p62 degradation, and lipid droplet dynamics upon GRAMD2A loss. This model thus enables dissection of GRAMD2A??s contribution to mTORC1-responsive autophagy and lysosomal cholesterol trafficking.
Representative applications include Western blotting for LC3-II and p62 under nutrient replete and starvation conditions, autophagy flux assays using lysosomal inhibitors or tandem fluorescent LC3 reporters, and cholesterol efflux measurements with fluorescent sterols. Lipid droplet accumulation can be monitored by Nile Red staining, and RT-qPCR can quantify changes in TFEB- and SREBP1-regulated gene networks. Co-immunoprecipitation validates disrupted ATG16L1/ BECN1 binding. The model is ideal for autophagy-modulator screening and metabolic research. Please contact Ascent Research for further details.
