APPL2 CRISPR Activation Plasmid (m2): sc-432068-ACT-2

Mouse Appl2 encodes APPL2, an endosomal adaptor protein containing BAR, PH, and PTB domains that links Rab5-positive early endosomes to signal transduction and membrane trafficking. APPL2 coordinates receptor-mediated endocytosis with downstream pathways including PI3K–AKT and MAPK signaling, influencing cytoskeletal dynamics, cell migration, and growth factor responses. Altered APPL family signaling has been associated with metabolic regulation, insulin sensitivity, and cancer-related signaling networks, making Appl2 a relevant target for studying endosome-dependent control of cellular homeostasis. Gene editing of Appl2 in mouse systems supports mechanistic dissection of endocytic scaffolding functions, pathway crosstalk, and genotype–phenotype relationships in cell biology and disease-model research.

APPL2 CRISPR Activation Plasmid (m2) provides a targeted, non-destructive approach to upregulating endogenous Appl2 expression without altering the underlying DNA sequence.

APPL2 CRISPR Activation Plasmid (m2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Appl2 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 Appl2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous APPL2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Appl2 locus and enabling the study of APPL2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of APPL2 pathway restoration in tumor cells with silenced or reduced Appl2 expression.

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