Nogo CRISPR Activation Plasmid (h): sc-400819-ACT

RTN4 encodes Nogo (also known as reticulon-4), an endoplasmic reticulum and myelin-associated protein that modulates neurite outgrowth, axon regeneration, and synaptic plasticity. Nogo isoforms signal through receptors such as NgR1 and co-receptors to influence RhoA/ROCK-dependent cytoskeletal remodeling, growth cone collapse, and neuron–glia interactions. In the CNS, Nogo contributes to activity-dependent circuit refinement and limits structural remodeling after injury, linking RTN4 regulation to pathways governing neuronal connectivity. Altered Nogo signaling and expression have been studied in contexts including neurotrauma, neurodegeneration, and neuropsychiatric phenotypes where axonal sprouting and network stability are perturbed.

Nogo CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RTN4 expression without altering the underlying DNA sequence.

Nogo CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RTN4 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 RTN4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Nogo expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RTN4 locus and enabling the study of Nogo-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Nogo pathway restoration in tumor cells with silenced or reduced RTN4 expression.

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