Flt-4 CRISPR Activation Plasmid (h): sc-400684-ACT

FLT4 encodes the human receptor tyrosine kinase Flt-4 (VEGFR-3), a principal signaling node in lymphatic endothelial cells that binds VEGF-C and VEGF-D to regulate lymphangiogenesis, vascular remodeling, and endothelial survival. Upon ligand engagement, Flt-4 activates downstream pathways including PI3K–AKT, MAPK/ERK, and PLCγ–PKC to coordinate proliferation, migration, and permeability responses. Dysregulated FLT4 signaling is implicated in abnormal lymphatic development and tissue fluid homeostasis and is frequently studied in the context of tumor-associated lymphangiogenesis and metastatic dissemination. As a membrane receptor with context-dependent signaling outputs, FLT4 serves as a tractable target for dissecting ligand–receptor dynamics, receptor trafficking, and pathway cross-talk in vascular biology.

Flt-4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous FLT4 expression without altering the underlying DNA sequence.

Flt-4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FLT4 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 FLT4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Flt-4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FLT4 locus and enabling the study of Flt-4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Flt-4 pathway restoration in tumor cells with silenced or reduced FLT4 expression.

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