Rab11-FIP5 CRISPR Activation Plasmid (h): sc-407779-ACT

RAB11FIP5 encodes Rab11-FIP5, a Rab11 family interacting protein that functions as an effector in Rab11-dependent recycling endosome trafficking. It coordinates vesicle sorting and membrane recycling processes that influence receptor turnover, endocytosis, and polarized transport, thereby shaping signaling outputs at the plasma membrane. Through its roles in endosomal dynamics and cargo recycling, Rab11-FIP5 contributes to regulation of cell migration, adhesion, and cytoskeletal organization. Dysregulated recycling pathways involving Rab11 effectors have been linked to altered signaling and invasive phenotypes observed across multiple disease-relevant models, supporting its utility in studies of trafficking-dependent mechanisms.

Rab11-FIP5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAB11FIP5 expression without altering the underlying DNA sequence.

Rab11-FIP5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAB11FIP5 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 RAB11FIP5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rab11-FIP5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAB11FIP5 locus and enabling the study of Rab11-FIP5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rab11-FIP5 pathway restoration in tumor cells with silenced or reduced RAB11FIP5 expression.

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