ALK CRISPR Activation Plasmid (h): sc-400460-ACT

ALK (anaplastic lymphoma kinase) encodes a receptor tyrosine kinase of the insulin receptor superfamily that regulates cell growth, survival, and neuronal development. Upon ligand-dependent activation and autophosphorylation, ALK propagates signaling through MAPK/ERK, PI3K–AKT, and JAK/STAT pathways, influencing transcriptional programs and cytoskeletal dynamics. In human disease biology, aberrant ALK activation via gene fusions, amplification, or activating point mutations is linked to oncogenic signaling networks and altered differentiation states. These properties make ALK a widely used node for studying receptor tyrosine kinase pathway wiring, signal transduction feedback, and context-dependent transcriptional responses.

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

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

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