MDC1 CRISPR Activation Plasmid (h): sc-405652-ACT

Mediator of DNA Damage Checkpoint 1 (MDC1) is a nuclear scaffold protein that amplifies DNA double-strand break signaling by binding phosphorylated H2AX (γH2AX) and coordinating the recruitment of repair and checkpoint factors. Through interactions that support ATM-dependent signaling, MDC1 promotes DNA damage checkpoint activation, chromatin remodeling at break sites, and pathway choice across homologous recombination and non-homologous end joining. By stabilizing damage foci and propagating ubiquitin-dependent signaling cascades, MDC1 contributes to genome integrity during replication stress and after genotoxic insult. Altered MDC1 function or expression has been associated with genomic instability phenotypes relevant to cancer biology and inherited defects in DNA damage response networks.

MDC1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MDC1 expression without altering the underlying DNA sequence.

MDC1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MDC1 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 MDC1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MDC1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MDC1 locus and enabling the study of MDC1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MDC1 pathway restoration in tumor cells with silenced or reduced MDC1 expression.

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