GALT CRISPR Activation Plasmid (h): sc-405717-ACT

Human GALT (galactose-1-phosphate uridylyltransferase) is a cytosolic enzyme in the Leloir pathway that catalyzes the conversion of galactose-1-phosphate and UDP-glucose to glucose-1-phosphate and UDP-galactose, supporting cellular carbohydrate utilization and UDP-sugar homeostasis. By maintaining balanced pools of galactose metabolites and nucleotide sugars, GALT influences glycosylation capacity and broader metabolic network stability. Disruption of GALT activity is classically associated with galactosemia phenotypes and provides a well-defined model for investigating metabolic stress responses and downstream effects on macromolecule biosynthesis. In experimental systems, modulation of GALT expression is used to interrogate galactose handling, UDP-sugar–dependent glycosylation pathways, and metabolic liabilities under defined nutrient conditions.

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

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

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