Myf-5 CRISPR Activation Plasmid (h): sc-400449-ACT

MYF5 encodes the myogenic regulatory factor Myf-5, a basic helix–loop–helix transcription factor that functions as an early determinant of skeletal muscle lineage commitment. Myf-5 coordinates myoblast specification and differentiation by regulating myogenic gene networks and cooperating with other MRF family members to control cell-cycle exit and muscle structural gene expression. Its activity integrates with developmental signaling inputs, including pathways such as Wnt and Notch, to shape progenitor fate decisions and muscle patterning. Dysregulated MYF5 expression or regulatory circuitry has been linked to altered myogenesis and is frequently studied in contexts such as sarcomas and other tumors exhibiting myogenic programs.

Myf-5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MYF5 expression without altering the underlying DNA sequence.

Myf-5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MYF5 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 MYF5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Myf-5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MYF5 locus and enabling the study of Myf-5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Myf-5 pathway restoration in tumor cells with silenced or reduced MYF5 expression.

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