The ANKRD54 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HEK293T cell line, engineered for targeted disruption of the ANKRD54 gene. This polyclonal pool provides a versatile loss-of-function model for studying the role of ANKRD54 in autophagy and related cellular processes. Using CRISPR/Cas9-mediated gene disruption, these cells enable researchers to investigate functional consequences of ANKRD54 ablation without clonal isolation, preserving genetic diversity within the population.
HEK293T cells are a widely used embryonic kidney epithelial cell line derived from HEK293, stably expressing the SV40 large T-antigen. This antigen facilitates episomal replication of plasmids containing the SV40 origin, making HEK293T cells a preferred host for transient protein expression, viral packaging, and large-scale protein production. Their robust growth and ease of transfection render them ideal for high-throughput screening and mechanistic studies.
ANKRD54 encodes an ankyrin repeat domain-containing adaptor protein that functions in selective autophagy, a cellular degradation pathway for specific cargo. The protein is activated by upstream signals such as nutrient deprivation, mTOR inhibition, and TFEB transcription factor activity. ANKRD54 interacts directly with members of the LC3/GABARAP family (including LC3A, LC3B, GABARAP, GABARAPL1, and GABARAPL2), which are ATG8-family ubiquitin-like proteins conjugated to autophagosomal membranes. By tethering selective autophagic cargo to these membrane proteins, ANKRD54 facilitates cargo engulfment and lysosomal degradation. This places ANKRD54 downstream of core autophagy initiation components like ULK1, ATG13, BECN1, and ATG5, and upstream of degradation effectors such as p62 and lysosomal enzymes. Disruption of ANKRD54 impairs the autophagic flux of specific substrates, potentially disrupting cellular homeostasis and stress responses.
In HEK293T cells, knockout of ANKRD54 creates a valuable system to interrogate selective autophagy mechanisms. HEK293T cells have a well-characterized proteome and autophagy machinery, and the polyclonal knockout population avoids clonal artifacts while offering a genetically perturbed background for functional assays. The absence of ANKRD54 in these cells can lead to accumulation of autophagy substrates and altered LC3-II turnover, providing measurable endpoints. This model is particularly useful for dissecting the role of selective autophagy in endolysosomal trafficking, since ANKRD54 bridges cargo recognition and autophagosome formation. It also permits the study of cross-talk between autophagy and other cellular pathways under conditions of nutrient stress or pharmacological mTOR inhibition.
These polyclonal knockout cells are suitable for a broad range of applications, including mechanistic studies of selective autophagy, drug screening for autophagy modulators, and protein interaction analyses. Common assays include western blotting to monitor LC3-II levels and substrate accumulation, immunofluorescence to visualize LC3 puncta and lysosomal markers, co-immunoprecipitation to identify ANKRD54 interactors, autophagy flux assays using chloroquine treatment, RT-qPCR for autophagy gene expression profiling, flow cytometry for cell cycle and apoptosis analysis, and electron microscopy for autophagosome quantification. For further information, please contact Ascent Research.
