GSC CRISPR Activation Plasmid (h): sc-404148-ACT

Human GSC (goosecoid homeobox) encodes a homeodomain transcription factor that regulates early embryonic patterning and mesendoderm specification by controlling gene expression programs linked to cell fate decisions and tissue morphogenesis. GSC participates in developmental transcriptional networks that integrate with signaling pathways such as TGF-β/SMAD and WNT/β-catenin, influencing epithelial–mesenchymal transition, migration, and differentiation states. Dysregulated GSC expression has been associated with altered developmental gene regulation and has been studied in contexts including tumor cell plasticity and invasive phenotypes, making it relevant for investigating transcriptional control of lineage identity. As a nuclear DNA-binding protein, GSC is commonly used as a marker and regulator in models of early development, stem cell differentiation, and EMT-associated transcriptional reprogramming.

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

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

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