DIO3 CRISPR Activation Plasmid (h): sc-403729-ACT

DIO3 encodes type III iodothyronine deiodinase (DIO3), an integral membrane selenoenzyme that inactivates thyroid hormones by converting T4 to reverse T3 and T3 to T2, thereby limiting local thyroid hormone signaling. By shaping tissue-specific hormone availability, DIO3 influences transcriptional programs governed by thyroid hormone receptors and contributes to developmental timing, metabolic homeostasis, and cellular differentiation. DIO3 expression is regulated in contexts such as genomic imprinting and hypoxia-associated signaling, and dysregulation has been linked to altered endocrine set points and growth control. In cancer and developmental disorders, aberrant DIO3 activity is frequently studied as a mechanism for modifying thyroid hormone–dependent gene expression networks within the tumor microenvironment or during organogenesis.

DIO3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous DIO3 expression without altering the underlying DNA sequence.

DIO3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the DIO3 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 DIO3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DIO3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native DIO3 locus and enabling the study of DIO3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DIO3 pathway restoration in tumor cells with silenced or reduced DIO3 expression.

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