Thymidine Kinase CRISPR Activation Plasmid (h): sc-402954-ACT

Human TK1 encodes thymidine kinase 1, a cytosolic enzyme that phosphorylates thymidine to thymidine monophosphate in the pyrimidine salvage pathway, supporting balanced dTTP pools required for DNA replication and repair. TK1 expression is tightly linked to S phase and proliferative status, integrating with cell-cycle regulation and nucleotide metabolism programs that influence genome stability. Altered TK1 activity and expression have been associated with dysregulated proliferation and DNA damage responses in cancer biology and other contexts where replication stress is prominent. As a readout and driver of nucleotide homeostasis, TK1 is frequently used to study replication dynamics, salvage pathway flux, and cell-cycle–dependent metabolic control.

Thymidine Kinase CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TK1 expression without altering the underlying DNA sequence.

Thymidine Kinase CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TK1 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 TK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Thymidine Kinase expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TK1 locus and enabling the study of Thymidine Kinase-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Thymidine Kinase pathway restoration in tumor cells with silenced or reduced TK1 expression.

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