ORP-8 CRISPR Activation Plasmid (h): sc-405318-ACT

OSBPL8 encodes oxysterol-binding protein-related protein 8 (ORP-8), a lipid-transfer and sterol-sensing factor that helps coordinate cellular cholesterol and phospholipid distribution at membrane contact sites, particularly between the endoplasmic reticulum and other organelles. ORP-8 contributes to regulation of lipid metabolism, membrane composition, and signaling processes linked to phosphoinositide handling and vesicular trafficking, shaping cellular homeostasis under changing nutrient or stress conditions. Altered ORP-8 activity has been studied in contexts involving dysregulated lipid handling and inflammatory signaling, which are frequently implicated in metabolic and cardiovascular disease biology as well as tumor-associated metabolic remodeling. In human cell models, ORP-8 modulation is therefore relevant for dissecting lipid-driven pathway crosstalk that influences proliferation, stress responses, and organelle communication.

ORP-8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous OSBPL8 expression without altering the underlying DNA sequence.

ORP-8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the OSBPL8 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 OSBPL8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ORP-8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native OSBPL8 locus and enabling the study of ORP-8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ORP-8 pathway restoration in tumor cells with silenced or reduced OSBPL8 expression.

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