APPL1 CRISPR Activation Plasmid (m): sc-428481-ACT

Mouse Appl1 encodes APPL1, an endosomal adaptor protein that couples Rab5-positive early endosome dynamics to signal transduction downstream of receptor tyrosine kinases. APPL1 participates in PI3K–AKT signaling, insulin and adiponectin responses, and modulation of MAPK and NF-κB-associated pathways through protein–protein interactions that coordinate trafficking with transcriptional outputs. Through these roles, APPL1 is broadly relevant to studies of metabolic homeostasis, insulin sensitivity, inflammation, and cellular growth control. Dysregulated APPL1-linked signaling and endocytic routing has been implicated in models of metabolic disease and altered proliferative signaling, making Appl1 a useful node for pathway interrogation in vivo and in cultured cells.

APPL1 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Appl1 expression without altering the underlying DNA sequence.

APPL1 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Appl1 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 Appl1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous APPL1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Appl1 locus and enabling the study of APPL1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of APPL1 pathway restoration in tumor cells with silenced or reduced Appl1 expression.

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