Retinal RX CRISPR Activation Plasmid (h): sc-403576-ACT

RAX (retinal homeobox) encodes the Retinal RX transcription factor, a paired-type homeodomain regulator essential for early eye field specification and retinal progenitor maintenance. It functions upstream of photoreceptor and neuroretinal differentiation programs by coordinating developmental gene regulatory networks and influencing cell fate decisions during neuroectoderm patterning. RAX activity interfaces with pathways that govern proliferation and differentiation in the developing retina, including signaling inputs that refine ocular morphogenesis. Dysregulated RAX expression or function is linked to congenital eye malformations such as anophthalmia/microphthalmia and broader retinal developmental defects, supporting its relevance in disease modeling and developmental biology research.

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

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

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