MAD2 CRISPR Activation Plasmid (h): sc-401027-ACT

Human MAD2L1 encodes MAD2, a core component of the spindle assembly checkpoint that monitors kinetochore–microtubule attachment and delays anaphase onset until chromosomes achieve bi-orientation. MAD2 participates in mitotic checkpoint complex formation to inhibit APC/C-CDC20 activity, thereby coordinating cyclin B and securin stability, chromosome segregation, and maintenance of genomic integrity. Dysregulation of MAD2L1 expression or checkpoint signaling can promote chromosomal instability and aneuploidy, features frequently observed in proliferative disease biology. As a result, MAD2L1 is widely studied in pathways controlling cell cycle progression, mitotic timing, and genome surveillance.

MAD2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MAD2L1 expression without altering the underlying DNA sequence.

MAD2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MAD2L1 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 MAD2L1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MAD2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MAD2L1 locus and enabling the study of MAD2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MAD2 pathway restoration in tumor cells with silenced or reduced MAD2L1 expression.

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