Med4 CRISPR Activation Plasmid (h): sc-406004-ACT

Human MED4 encodes Med4, a conserved subunit of the Mediator complex that links sequence-specific transcription factors to RNA polymerase II to coordinate transcription initiation. Med4 contributes to transcriptional programs that govern cell-cycle progression, differentiation, and stress-responsive gene expression through broad regulation of Pol II–dependent transcription. Perturbation of Mediator subunits can shift global transcriptional output, with downstream effects on proliferation, genome stability, and lineage-specific regulatory networks that are frequently altered in cancer and other disorders involving transcriptional dysregulation. As a node in transcriptional control, MED4 is useful for studying how Mediator architecture tunes promoter activation and signaling-dependent gene expression.

Med4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MED4 expression without altering the underlying DNA sequence.

Med4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MED4 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 MED4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Med4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MED4 locus and enabling the study of Med4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Med4 pathway restoration in tumor cells with silenced or reduced MED4 expression.

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