MLK2 CRISPR Activation Plasmid (h): sc-406968-ACT

Human MAP3K10 encodes mixed lineage kinase 2 (MLK2), a MAP kinase kinase kinase that integrates upstream cues from small GTPases and stress signals to propagate phosphorylation cascades. MLK2 is a key regulator of MAPK signaling, particularly JNK and p38 pathways, shaping transcriptional programs that control apoptosis, inflammatory responses, and neuronal differentiation. Through these signaling nodes, MLK2 influences cytoskeletal dynamics and cellular stress adaptation, processes frequently interrogated in cancer biology and neurodegeneration research. Dysregulated MAP3K10 activity has been associated with aberrant stress-activated signaling and altered survival pathways, making it relevant for mechanistic studies of pathway rewiring.

MLK2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MAP3K10 expression without altering the underlying DNA sequence.

MLK2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MAP3K10 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 MAP3K10 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MLK2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MAP3K10 locus and enabling the study of MLK2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MLK2 pathway restoration in tumor cells with silenced or reduced MAP3K10 expression.

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