FXYD3 CRISPR Activation Plasmid (h2): sc-406431-ACT-2

Human FXYD3 encodes a small single-pass membrane regulator of Na⁺/K⁺-ATPase, modulating pump kinetics and thereby influencing cellular ion homeostasis, membrane potential, and volume regulation in epithelial contexts. Through its control of sodium and potassium transport, FXYD3 impacts downstream processes linked to proliferation, differentiation, and stress adaptation that intersect with signaling networks sensitive to ionic balance. Altered FXYD3 expression has been reported across multiple carcinomas and other epithelial pathologies, supporting its relevance for studies of dysregulated transport biology and tumor-associated metabolic and microenvironmental changes. Gene editing or perturbation of FXYD3 enables mechanistic investigation of Na⁺/K⁺-ATPase regulation, membrane proteostasis, and ion-dependent phenotypes in human cell models, including functional assays of pump activity, migration, and epithelial barrier properties.

FXYD3 CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous FXYD3 expression without altering the underlying DNA sequence.

FXYD3 CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FXYD3 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 FXYD3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous FXYD3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FXYD3 locus and enabling the study of FXYD3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of FXYD3 pathway restoration in tumor cells with silenced or reduced FXYD3 expression.

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