Aquaporin 0/AQP0 CRISPR Activation Plasmid (h): sc-403852-ACT

MIP encodes aquaporin 0 (AQP0), the predominant membrane intrinsic protein of lens fiber cells that functions as a low-permeability water channel and an adhesion molecule supporting tight fiber–fiber apposition. By regulating lens water homeostasis and contributing to membrane organization, AQP0 helps maintain lens transparency and refractive properties through processes linked to osmotic balance, junctional architecture, and proteostasis. Its activity is influenced by post-translational modifications and interactions with lens cytoskeletal and junctional components that shape fiber cell maturation. Dysregulation or mutation of MIP is associated with inherited cataracts and lens microarchitecture defects, making it relevant for studies of lens development, protein aggregation, and barrier physiology.

Aquaporin 0/AQP0 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MIP expression without altering the underlying DNA sequence.

Aquaporin 0/AQP0 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MIP 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 MIP transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Aquaporin 0/AQP0 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MIP locus and enabling the study of Aquaporin 0/AQP0-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Aquaporin 0/AQP0 pathway restoration in tumor cells with silenced or reduced MIP expression.

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