Nucleostemin CRISPR Activation Plasmid (h): sc-402271-ACT

Human GNL3 encodes nucleostemin, a nucleolar GTP-binding protein enriched in stem and progenitor populations that helps coordinate ribosome biogenesis with cell-cycle progression. Nucleostemin participates in nucleolar stress signaling and interfaces with p53- and MDM2-associated checkpoints to modulate proliferation, senescence, and genome stability. Through these processes it contributes to regulation of self-renewal, mitotic fidelity, and cellular responses to oncogenic or replication stress. Altered GNL3 expression has been associated with dysregulated growth programs in multiple tumor contexts and is frequently used as a marker for proliferative cell states in biomedical research.

Nucleostemin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GNL3 expression without altering the underlying DNA sequence.

Nucleostemin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GNL3 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 GNL3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Nucleostemin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GNL3 locus and enabling the study of Nucleostemin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Nucleostemin pathway restoration in tumor cells with silenced or reduced GNL3 expression.

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