Amylase CRISPR Activation Plasmid (h): sc-400533-ACT

AMY2A encodes a secreted pancreatic α-amylase that initiates carbohydrate digestion by hydrolyzing α-1,4-glycosidic bonds in dietary starch and glycogen to generate maltose and oligosaccharides. Its expression and secretion are tightly linked to exocrine pancreatic differentiation and regulated secretory pathway biology, integrating nutrient-responsive transcriptional programs with zymogen granule trafficking. Altered amylase output is commonly used as a functional readout of pancreatic acinar state and digestive enzyme homeostasis, and AMY2A is studied in contexts where exocrine function is perturbed, including pancreatic inflammation and broader digestive enzyme dysregulation. As a highly expressed secreted enzyme, AMY2A also serves as a model locus for investigating transcriptional control of secreted proteins and acinar cell identity.

Amylase 2a CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AMY2A expression without altering the underlying DNA sequence.

Amylase 2a CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AMY2A 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 AMY2A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Amylase 2a expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AMY2A locus and enabling the study of Amylase 2a-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Amylase 2a pathway restoration in tumor cells with silenced or reduced AMY2A expression.

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