The ATG7 Knockout HaCaT Cell Line is a CRISPR/Cas9-edited human keratinocyte line with targeted disruption of the ATG7 gene. This knockout model eliminates the E1-like ubiquitin-activating enzyme essential for both major ubiquitin-like conjugation systems in autophagy. As a result, the cell line is incapable of forming autophagosomes, providing a genetically stable tool for studying autophagy-dependent processes. It serves as a robust platform for dissecting ATG7-mediated mechanisms in epidermal biology, enabling detailed analysis of pathway functions and substrates in a well-characterized keratinocyte background.
HaCaT cells are a spontaneously immortalized, non-tumorigenic human keratinocyte line widely used to model epidermal homeostasis, differentiation, and barrier function. Retaining many features of normal keratinocytes, they express differentiation markers such as involucrin and keratin 10 and can form stratified cultures. Their genetic stability and extensive characterization make HaCaT an ideal host for studying autophagy in the context of skin barrier maintenance, wound healing, and dermatological disorders like psoriasis and impaired re-epithelialization.
ATG7 is the E1-like enzyme for autophagy??s two ubiquitin-like conjugation systems. It catalyzes ATP-dependent activation of ATG12 for conjugation to ATG5 by ATG10, and activates LC3 proteins for lipidation by ATG3, steps essential for autophagosome expansion. Upstream, mTORC1, AMPK, and transcription factors TFEB and FOXO3 regulate ATG7 in response to nutrient status. Downstream, ATG7 is required for forming the ATG12?CATG5?CATG16L1 complex and generating LC3-II. In the autophagy pathway, ATG7 operates downstream of the ULK1 and Beclin1?CVPS34 complexes, interacting with ATG3, ATG10, and ATG5. Knockout blocks autophagic flux, causing accumulation of p62/SQSTM1 and damaged organelles.
In keratinocytes, autophagy is critical for epidermal barrier formation, differentiation, and stress responses. The ATG7 Knockout HaCaT Cell Line provides a relevant model to study these processes. Loss of ATG7 impairs autophagosome formation, leading to compromised barrier integrity, altered differentiation, and defective wound healing. Dysregulated autophagy is implicated in skin disorders such as psoriasis and chronic wounds, making this knockout line valuable for investigating disease mechanisms and potential therapeutic interventions.
This ATG7 knockout line enables a broad range of autophagy and keratinocyte biology studies. Researchers can perform western blotting to confirm ATG7 deletion and monitor LC3-II and p62 levels, immunofluorescence to visualize LC3 puncta, and autophagic flux assays using bafilomycin A1. Functional assays include cell viability measurements under nutrient starvation, RT-qPCR for differentiation markers like involucrin and keratin 10, and wound healing scratch assays. The model also facilitates drug resistance/sensitivity screening and proliferation analyses. For further details, contact Ascent Research.
