Apg-1 CRISPR Activation Plasmid (h): sc-403942-ACT

HSPA4L encodes Apg-1, a member of the HSP70 family of ATP-dependent molecular chaperones that supports proteostasis by promoting client protein folding, preventing aggregation, and facilitating recovery from cellular stress. Apg-1 is implicated in cytoprotective heat shock responses and coordination of quality-control pathways linked to ubiquitin–proteasome turnover and autophagy, helping maintain protein homeostasis during oxidative, thermal, and ER stress. As proteostasis imbalance is a common feature of cancer, neurodegeneration, and other stress-associated pathologies, altered HSPA4L activity is studied in contexts where chaperone capacity, protein misfolding, and stress adaptation influence cell fate decisions. Human Apg-1 also provides a useful model for dissecting HSP70-network crosstalk with signaling nodes that couple stress sensing to apoptosis, cell cycle control, and metabolic remodeling.

Apg-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HSPA4L expression without altering the underlying DNA sequence.

Apg-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HSPA4L 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 HSPA4L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Apg-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HSPA4L locus and enabling the study of Apg-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Apg-1 pathway restoration in tumor cells with silenced or reduced HSPA4L expression.

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