GBX2 CRISPR Activation Plasmid (m): sc-420503-ACT

Mouse Gbx2 encodes the homeobox transcription factor GBX2, a key regulator of embryonic patterning and regional identity in the developing central nervous system. GBX2 functions in transcriptional programs that coordinate neural tube and hindbrain development, interacting with morphogen-driven networks such as WNT, FGF, and retinoic acid signaling to establish anterior–posterior boundaries and neuronal lineage specification. Perturbation of GBX2-dependent gene regulation is relevant to studies of neurodevelopmental dysregulation and congenital malformations, where altered boundary formation and cell fate decisions can have lasting phenotypic consequences. In adult and stem/progenitor contexts, GBX2 is used as a marker and mechanistic node for investigating differentiation trajectories and transcription factor hierarchies.

GBX2 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Gbx2 expression without altering the underlying DNA sequence.

GBX2 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Gbx2 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 Gbx2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GBX2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Gbx2 locus and enabling the study of GBX2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GBX2 pathway restoration in tumor cells with silenced or reduced Gbx2 expression.

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