KIAA0232 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population of human HEK293T cells with targeted disruption of the KIAA0232 gene. This heterogeneous knockout model, generated by CRISPR/Cas9-mediated gene ablation, provides a loss-of-function tool for studying autophagy and membrane trafficking without clonal selection artifacts. The polyclonal pool enables robust functional assays while representing a range of edited alleles.
HEK293T cells are adherent human embryonic kidney epithelial cells expressing SV40 large T antigen, offering high transfection efficiency and utility for transient expression, viral packaging, and recombinant protein production. Their well-characterized signaling and ease of genetic manipulation make them a standard model for gene-editing studies, particularly in autophagy research where protocols for flux assays and LC3 analysis are well established.
The KIAA0232 gene is predicted to encode a TBC domain-containing Rab GTPase-activating protein (GAP) that regulates macroautophagy by controlling Rab activity during autophagosome formation and maturation. KIAA0232 functions downstream of mTOR nutrient signaling and the ULK1 complex, interacting with core autophagy proteins including ATG14, Beclin-1, VPS34, and ATG16L1. It is thought to modulate RAB7 and RAB11 activity, influencing ATG5-ATG12 conjugation and LC3 lipidation. Disruption of KIAA0232 therefore impairs autophagic flux, leading to accumulation of autophagy substrates like p62 and altered LC3 processing.
In the HEK293T background, this knockout model enables investigation of Rab GAP function in autophagy degradation pathways. The polyclonal design captures diverse editing outcomes, reducing clonal bias, and the cells exhibit robust stress-inducible autophagy, making them suitable for nutrient-deprivation and pharmacological studies. Loss of KIAA0232 likely dysregulates Rab cycling, providing a system to dissect the crosstalk between Rab GTPases and the autophagic machinery in an epithelial context relevant to cancer and neurodegeneration.
Research applications include monitoring autophagy markers via Western blot (LC3-I/II, p62), immunofluorescence of LC3 puncta, and autophagic flux assays with Bafilomycin A1. The cells support co-immunoprecipitation of autophagy complexes and RT-qPCR of ATG genes, as well as drug target validation for autophagy modulators. They are particularly valuable for advancing studies in cancer cell biology, neurodegeneration, and membrane trafficking dynamics. For further information or support, contact Ascent Research.
