UKHC CRISPR Activation Plasmid (h): sc-402349-ACT

KIF5B encodes the kinesin-1 heavy chain isoform UKHC, a microtubule-based motor that powers ATP-dependent anterograde transport of vesicles, protein complexes, and organelles in polarized cells. Through coordination with kinesin light chains and cargo adaptors, KIF5B supports intracellular trafficking, centrosome and spindle dynamics, and cytoskeletal organization, thereby influencing processes such as mitosis, secretion, and neuronal transport. Dysregulated KIF5B activity or expression can perturb organelle distribution and signaling, and KIF5B is frequently studied in the context of oncogenic gene fusions (e.g., KIF5B-RET) and altered proliferative pathways. These features make KIF5B a useful node for investigating microtubule transport mechanisms, cell-cycle control, and trafficking-dependent signaling in human cell models.

UKHC CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KIF5B expression without altering the underlying DNA sequence.

UKHC CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KIF5B 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 KIF5B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UKHC expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KIF5B locus and enabling the study of UKHC-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UKHC pathway restoration in tumor cells with silenced or reduced KIF5B expression.

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