The ALOX12B Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from human embryonic kidney HEK293T cells. This model introduces targeted disruption of ALOX12B, creating a loss-of-function system for studying this epidermal-type lipoxygenase. The polyclonal pool captures diverse edited alleles, facilitating robust functional analyses without clonal selection. It is designed for investigating arachidonic acid metabolism and skin barrier formation, enabling definitive loss-of-function studies in the context of epidermal lipid signaling.
The HEK293T host cell line is a derivative of HEK293 cells that constitutively expresses SV40 large T antigen, enabling episomal replication of SV40 origin-containing plasmids and high-level transient protein expression. These embryonic kidney epithelial cells are extensively employed for heterologous protein production and transfection-based experiments due to their rapid growth, ease of culture, exceptional transfectability, and well-characterized proteome, making them an ideal chassis for studying gene function.
ALOX12B encodes an epidermis-type lipoxygenase that stereospecifically converts arachidonic acid to 12R-HETE. This reaction is central to epidermal lipid metabolism: 12R-HETE acts downstream with ALOXE3 to process omega-hydroxyceramides, which are incorporated into the lipid lamellae of the cornified envelope, forming the skin barrier. This process is essential for the formation of the lipid-bound cornified envelope, critical for preventing transepidermal water loss. ALOX12B is regulated by calcium and PPAR ligands and functions alongside CYP4F22 and ceramide synthase. Pathway disruption causes autosomal recessive congenital ichthyosis.
HEK293T cells are renal in origin and do not naturally undergo epidermal differentiation, yet they provide a clean heterologous system for reconstituting ALOX12B activity. This model permits isolated study of the lipoxygenase??s enzymatic function and its immediate products, independent of keratinocyte-specific programs. Transfection-based reconstitution of pathway components enables precise structure-function mapping of ceramide processing and mutagenesis of the catalytic domain.
These polyclonal knockout cells support diverse applications: ichthyosis disease modeling via pathway reconstitution, arachidonic acid metabolism studies, and LC-MS lipidomics for quantifying 12R-HETE and ceramides. They are compatible with western blotting, RT-qPCR, and enzymatic assays to confirm gene disruption and screen compounds for barrier disorder therapy. For further information, contact Ascent Research.
