CH25H CRISPR Activation Plasmid (m): sc-419641-ACT

Ch25h encodes cholesterol 25-hydroxylase (CH25H), an endoplasmic reticulum-associated enzyme that converts cholesterol to 25-hydroxycholesterol, a bioactive oxysterol that modulates sterol homeostasis and membrane lipid composition. Through regulation of SREBP-driven cholesterol biosynthesis and LXR-dependent transcriptional programs, CH25H influences lipid metabolism, vesicular trafficking, and inflammatory signaling. In mouse immune cells, Ch25h is inducible by innate immune stimuli and can shape macrophage and microglial activation states by altering oxysterol-mediated signaling networks. Dysregulated CH25H activity and 25-hydroxycholesterol production have been linked to aberrant inflammatory responses and metabolic stress pathways, making Ch25h a useful node for studying immunometabolic crosstalk in disease-relevant models.

CH25H CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Ch25h expression without altering the underlying DNA sequence.

CH25H CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ch25h 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 Ch25h transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CH25H expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ch25h locus and enabling the study of CH25H-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CH25H pathway restoration in tumor cells with silenced or reduced Ch25h expression.

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