DD4 CRISPR Activation Plasmid (h): sc-403692-ACT

Human AKR1C4 encodes DD4, an NADPH-dependent aldo-keto reductase that catalyzes the reduction of steroid hormones and reactive carbonyl substrates. DD4 functions prominently in hepatic steroid and bile acid metabolism, contributing to detoxification and redox homeostasis through regulation of ketosteroid interconversion. By modulating levels of androgens, progestins, and other steroid intermediates, AKR1C4 influences hormone-dependent transcriptional programs and metabolic signaling networks. Altered AKR1C4 expression or activity has been associated with dysregulated steroid metabolism and hepatic pathophysiology, supporting its relevance in studies of metabolic disease biology and xenobiotic response.

DD4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AKR1C4 expression without altering the underlying DNA sequence.

DD4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AKR1C4 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 AKR1C4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DD4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AKR1C4 locus and enabling the study of DD4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DD4 pathway restoration in tumor cells with silenced or reduced AKR1C4 expression.

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