MLKL CRISPR Activation Plasmid (h): sc-401248-ACT

Human MLKL (mixed lineage kinase domain-like protein) is a core executioner of necroptosis, acting downstream of RIPK1/RIPK3 signaling to drive inflammatory, lytic cell death. Upon phosphorylation by RIPK3, MLKL undergoes conformational activation, oligomerizes, and translocates to cellular membranes where it perturbs membrane integrity and promotes release of damage-associated molecular patterns. This pathway intersects with innate immune responses, cytokine signaling, and regulated cell death crosstalk with apoptosis and pyroptosis, shaping tissue inflammation and host defense. Dysregulated MLKL activity and necroptotic signaling have been implicated in models of inflammatory and neurodegenerative processes, ischemic injury, and tumor microenvironment biology, making MLKL a useful node for mechanistic studies.

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

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

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