VPS26B CRISPR Activation Plasmid (h): sc-410249-ACT

VPS26B encodes a core component of the retromer complex that governs endosomal cargo sorting and recycling from endosomes to the trans-Golgi network and plasma membrane. Through coordination with VPS35 and VPS29, VPS26B helps maintain membrane protein homeostasis, regulates receptor trafficking, and supports lysosomal function by limiting aberrant endosome maturation. Retromer-dependent pathways influence cell signaling, nutrient transporter localization, and intracellular proteostasis, linking VPS26B activity to broader processes such as autophagy and endolysosomal stress responses. Dysregulation of endosomal trafficking and retromer function has been implicated in neurodegenerative and developmental phenotypes, making VPS26B a relevant target for mechanistic studies of membrane trafficking–associated disease biology.

VPS26B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS26B expression without altering the underlying DNA sequence.

VPS26B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS26B 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 VPS26B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS26B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS26B locus and enabling the study of VPS26B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS26B pathway restoration in tumor cells with silenced or reduced VPS26B expression.

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