EHD3 CRISPR Activation Plasmid (h2): sc-404685-ACT-2

Human EHD3 (EH domain-containing protein 3) is an ATPase of the EHD family that regulates endocytic recycling and membrane tubulation, coordinating trafficking from early endosomes to recycling endosomes and back to the plasma membrane. By interacting with Rab effectors and membrane-associated partners, EHD3 influences receptor internalization and recycling, vesicle scission, and spatial organization of signaling platforms that shape cell polarity and migration. Altered EHD3 expression has been linked to dysregulated endosomal transport programs observed in cancer biology and in neurological contexts where membrane recycling impacts synaptic function and cellular homeostasis. Gene editing of EHD3 in human cell models enables mechanistic studies of endosome-to-membrane trafficking, receptor fate decisions, and downstream signaling consequences using imaging, proteomics, and functional assays of vesicular dynamics.

EHD3 CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous EHD3 expression without altering the underlying DNA sequence.

EHD3 CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EHD3 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 EHD3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EHD3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EHD3 locus and enabling the study of EHD3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EHD3 pathway restoration in tumor cells with silenced or reduced EHD3 expression.

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