TPO CRISPR Activation Plasmid (h): sc-417243-ACT

Human THPO encodes thrombopoietin (TPO), a secreted cytokine that is the principal regulator of megakaryocyte proliferation and platelet production. TPO signals primarily through MPL to activate JAK2/STAT5, PI3K/AKT, and MAPK pathways, supporting hematopoietic stem and progenitor cell maintenance and lineage commitment. Dysregulated THPO–MPL signaling has been linked to altered platelet homeostasis and contributes to the pathobiology of myeloproliferative neoplasms and other disorders of hematopoiesis. As a liver-predominant endocrine factor with feedback control by platelet mass, THPO provides a tractable node for studying cytokine-driven transcriptional programs and hematopoietic niche signaling.

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

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

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