COL4A4 CRISPR Activation Plasmid (h): sc-404186-ACT

COL4A4 encodes the collagen type IV alpha 4 chain, a core structural component of basement membranes that assembles into heterotrimeric type IV collagen networks supporting epithelial and endothelial interfaces. These networks regulate extracellular matrix organization, cell adhesion, polarity, and tissue barrier function, influencing signaling processes such as integrin-mediated pathways and matrix-dependent mechanotransduction. In the kidney glomerular basement membrane, COL4A4 contributes to filtration architecture and matrix stability, and altered COL4A4 function is linked to inherited nephropathies including Alport spectrum disorders and thin basement membrane phenotypes. Dysregulation of type IV collagen composition also impacts vascular and epithelial microenvironments relevant to fibrosis and tissue remodeling research.

COL4A4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous COL4A4 expression without altering the underlying DNA sequence.

COL4A4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the COL4A4 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 COL4A4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous COL4A4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native COL4A4 locus and enabling the study of COL4A4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of COL4A4 pathway restoration in tumor cells with silenced or reduced COL4A4 expression.

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