RyR-3 CRISPR Activation Plasmid (m2): sc-422776-ACT-2

Mouse Ryr3 encodes the ryanodine receptor 3 (RyR-3), an intracellular Ca2+ release channel primarily localized to the endoplasmic/sarcoplasmic reticulum that mediates calcium-induced calcium release and shapes cytosolic calcium transients. RyR-3-dependent Ca2+ signaling contributes to excitation–contraction coupling, synaptic plasticity, and activity-dependent gene regulation by modulating processes such as calcium homeostasis, mitochondrial coupling, and downstream CaMK/CREB-linked pathways. Altered RyR-3 function and ryanodine receptor–mediated calcium leak have been implicated in neurodevelopmental and neurodegenerative phenotypes, seizure susceptibility, and muscle physiology defects in model systems. Targeted editing of Ryr3 in mouse supports mechanistic studies of intracellular calcium dynamics, electrophysiology, and ER stress responses, enabling functional validation in neuronal and muscle cell models and in vivo genetic circuits.

RyR-3 CRISPR Activation Plasmid (m2) provides a targeted, non-destructive approach to upregulating endogenous Ryr3 expression without altering the underlying DNA sequence.

RyR-3 CRISPR Activation Plasmid (m2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ryr3 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 Ryr3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RyR-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ryr3 locus and enabling the study of RyR-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RyR-3 pathway restoration in tumor cells with silenced or reduced Ryr3 expression.

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