MBD4 CRISPR Activation Plasmid (h): sc-403156-ACT

Human MBD4 (methyl-CpG binding domain protein 4) is a DNA glycosylase that recognizes T:G and U:G mismatches arising from deamination at methylated CpG sites and initiates base excision repair to maintain genome integrity. By coupling methyl-CpG recognition to lesion excision, MBD4 links epigenetic context with DNA repair and contributes to suppression of mutation accumulation at CpG dinucleotides. MBD4 activity intersects with replication-associated repair and damage response processes that shape mutation spectra and chromosomal stability. Altered MBD4 function or expression has been associated with hypermutation phenotypes and genomic instability observed in diverse cancer-relevant contexts, supporting its utility in studying mutagenesis mechanisms.

MBD4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MBD4 expression without altering the underlying DNA sequence.

MBD4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MBD4 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 MBD4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MBD4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MBD4 locus and enabling the study of MBD4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MBD4 pathway restoration in tumor cells with silenced or reduced MBD4 expression.

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