Ob CRISPR Activation Plasmid (h): sc-400706-ACT

Human LEP encodes the adipokine leptin (Ob), a key endocrine regulator of energy balance that communicates adipose nutrient status to the hypothalamus to modulate appetite and energy expenditure. Leptin signaling is mediated primarily through LEPR-driven JAK2/STAT3 activation and intersects with PI3K–AKT and MAPK pathways, influencing glucose homeostasis, lipid metabolism, and neuroendocrine function. In peripheral tissues, leptin also shapes immune and inflammatory processes, linking metabolic state to cytokine signaling and immune cell activity. Dysregulated LEP expression or leptin resistance is associated with obesity-related metabolic phenotypes and broader cardiometabolic and inflammatory disease biology, making LEP a central node for mechanistic studies.

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

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

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