5-LO CRISPR Activation Plasmid (h): sc-401239-ACT

Human ALOX5 encodes 5-lipoxygenase (5-LO), a non-heme iron dioxygenase that catalyzes the conversion of arachidonic acid into leukotriene intermediates, including 5-HPETE and leukotriene A4. This enzymatic step sits at the core of leukotriene biosynthesis and coordinates inflammatory lipid mediator signaling through autocrine and paracrine pathways in myeloid cells and other leukocyte populations. 5-LO activity interfaces with eicosanoid network crosstalk, calcium-dependent activation, and subcellular localization dynamics that shape downstream cytokine and chemotactic responses. Dysregulated ALOX5/5-LO signaling has been associated with inflammatory disease biology and immune microenvironment remodeling, making it a relevant node for mechanistic studies of immunometabolism and lipid signaling.

5-LO CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ALOX5 expression without altering the underlying DNA sequence.

5-LO CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ALOX5 locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the ALOX5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous 5-LO expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ALOX5 locus and enabling the study of 5-LO-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of 5-LO pathway restoration in tumor cells with silenced or reduced ALOX5 expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.