VPS35 CRISPR Activation Plasmid (h): sc-402019-ACT

VPS35 encodes a core subunit of the retromer complex that governs endosome-to-trans-Golgi network retrieval and endosomal recycling of diverse cargo proteins, thereby maintaining membrane protein homeostasis and organelle integrity. Through interactions with sorting nexins and the WASH complex, VPS35 helps coordinate endosomal tubulation, actin dynamics, and trafficking routes that influence receptor signaling, lysosomal function, and autophagy. Perturbation of VPS35-dependent sorting can alter mitochondrial quality control and proteostasis, linking retromer dysfunction to neurodegeneration and other disorders associated with defective endolysosomal trafficking. As a central node in intracellular transport pathways, VPS35 is broadly used to interrogate mechanisms of cargo sorting, synaptic maintenance, and stress responses in human cell models.

VPS35 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS35 expression without altering the underlying DNA sequence.

VPS35 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS35 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 VPS35 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS35 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS35 locus and enabling the study of VPS35-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS35 pathway restoration in tumor cells with silenced or reduced VPS35 expression.

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