KXD1 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the KXD1 gene in the human embryonic kidney cell line HEK293T. This loss-of-function model allows systematic investigation of KXD1-dependent signaling without clonal artefacts. The heterogeneous polyclonal pool ensures robust gene disruption across the population, making it suitable for bulk assays such as biochemical fractionation and proteomic analyses.
HEK293T cells originate from human embryonic kidney cells transformed with adenovirus type 5 DNA, conferring high transfectability and rapid growth. They stably express SV40 large T antigen, enhancing episomal replication and recombinant protein yields from SV40-ori vectors. These epithelial cells are widely used for protein production, lentiviral packaging, and functional genomics. Notably, HEK293T cells display robust basal mTORC1 activity, making them ideal for studying negative regulators like KICSTOR.
KXD1 is a core subunit of the KICSTOR complex, which also contains KPTN, ITFG2, and C12orf66. This lysosome-anchored complex recruits the GATOR1 complex (DEPDC5, NPRL2, NPRL3) upon amino acid starvation, exerting GAP activity toward Rag GTPases to maintain their inactive GDP-bound state. This prevents mTORC1 lysosomal recruitment and activation, suppressing phosphorylation of S6K and 4E-BP1 while triggering autophagy via ULK1 and TFEB. Upstream, both amino acid deprivation and AMPK signaling promote KICSTOR?CGATOR1-mediated mTORC1 inhibition.
In HEK293T cells, KXD1 loss disrupts the KICSTOR complex, unleashing constitutive mTORC1 activation that mimics the molecular phenotype of KXD1-linked neurodevelopmental disorders featuring macrocephaly, epilepsy, and brain malformations. This polyclonal knockout population thus serves as a tractable model for mTORC1 hyperactivity and its consequences, including dysregulated protein synthesis and blocked autophagy. Owing to HEK293T cells?? high transfectability, this background can be combined with cDNA rescue or mutant constructs to map KICSTOR structure?Cfunction relationships.
These polyclonal knockout cells are ideal for investigating mTORC1 signaling via phospho-S6K immunoblotting under full and amino acid?Cstarved conditions. Autophagy flux can be measured by LC3-II accumulation assays, while TFEB nuclear translocation is visualized by immunofluorescence. Co-immunoprecipitation experiments probe residual KICSTOR complex formation. The model supports screening of mTORC1 inhibitors and autophagy activators, and it is applicable to mTORopathy research. For product details and technical support, please contact Ascent Research.
