GLT25D2 CRISPR Activation Plasmid (h): sc-405276-ACT

COLGALT2 encodes GLT25D2, a collagen beta(1-O)galactosyltransferase that initiates glycosylation of hydroxylysine residues on collagen by adding galactose, a prerequisite for subsequent glucosylation and proper extracellular matrix maturation. This post-translational modification influences collagen folding, secretion, fibrillogenesis, and matrix organization, linking GLT25D2 activity to proteostasis within the secretory pathway and tissue-specific matrix remodeling. Altered collagen glycosylation is relevant to studies of fibrosis, connective tissue integrity, tumor-associated stromal dynamics, and musculoskeletal biology where extracellular matrix composition modulates signaling and mechanics. GLT25D2 is therefore a useful node for interrogating how collagen processing impacts cell–matrix interactions, adhesion, and downstream pathways responsive to matrix architecture.

GLT25D2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous COLGALT2 expression without altering the underlying DNA sequence.

GLT25D2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the COLGALT2 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 COLGALT2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GLT25D2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native COLGALT2 locus and enabling the study of GLT25D2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GLT25D2 pathway restoration in tumor cells with silenced or reduced COLGALT2 expression.

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