TNAP CRISPR Activation Plasmid (h): sc-400784-ACT

ALPL encodes tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme that hydrolyzes extracellular phosphate monoesters to regulate inorganic phosphate/pyrophosphate balance. By controlling pyrophosphate availability and generating phosphate for hydroxyapatite formation, TNAP is a central regulator of mineralization programs and osteoblast differentiation, and it interfaces with purinergic signaling through metabolism of nucleotides such as ATP. TNAP activity also influences extracellular matrix composition and calcification propensity across bone and vascular tissues. Dysregulated ALPL/TNAP function is linked to abnormal mineral deposition and skeletal phenotypes, making it relevant to studies of bone biology, calcification pathways, and phosphate homeostasis.

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

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

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