GlyT2 CRISPR Activation Plasmid (h): sc-403077-ACT

Human SLC6A5 encodes glycine transporter 2 (GlyT2), a presynaptic Na⁺/Cl⁻-dependent transporter that mediates high-affinity glycine reuptake into inhibitory glycinergic neurons. By sustaining vesicular glycine supply and shaping synaptic glycine availability, GlyT2 is central to neurotransmitter recycling, spinal cord and brainstem inhibitory circuitry, and overall regulation of motor and sensory processing. SLC6A5 function interfaces with solute carrier transport, ion-coupled membrane transport, and synaptic transmission pathways that influence neuronal excitability. Altered GlyT2 activity or expression is associated with dysregulated inhibitory neurotransmission and is relevant to research on inherited startle phenotypes and other disorders of motor control.

GlyT2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC6A5 expression without altering the underlying DNA sequence.

GlyT2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC6A5 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 SLC6A5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GlyT2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC6A5 locus and enabling the study of GlyT2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GlyT2 pathway restoration in tumor cells with silenced or reduced SLC6A5 expression.

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