Emt CRISPR Activation Plasmid (h): sc-402628-ACT

Human ITK encodes IL-2–inducible T-cell kinase, a Tec family non-receptor tyrosine kinase that couples T-cell receptor engagement to downstream signaling required for lymphocyte activation and differentiation. ITK participates in proximal TCR pathways that converge on PLCγ1 activation, calcium flux, MAPK signaling, and transcriptional programs controlling cytokine production and effector function. Dysregulated ITK activity is linked to altered Th1/Th2 balance, impaired antiviral immunity, and abnormal lymphoproliferation, making it relevant to studies of immune homeostasis and inflammatory disease mechanisms. As the Emt protein context, ITK provides a tractable node for dissecting signal transduction dynamics in human T cells and related hematopoietic lineages.

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

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

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