Erythropoietin/EPO CRISPR Activation Plasmid (h): sc-400283-ACT

EPO encodes erythropoietin, a secreted glycoprotein hormone that regulates erythroid progenitor survival, proliferation, and differentiation primarily through EPOR engagement and downstream JAK2/STAT5 signaling, with additional inputs from PI3K/AKT and MAPK pathways. Its expression is tightly controlled by oxygen-sensing mechanisms, notably HIF-dependent transcriptional regulation, coupling hypoxic stress to erythropoiesis. Dysregulated EPO/EPOR signaling and altered oxygen-response programs are relevant to anemia biology, polycythemia, and tumor hypoxia-associated adaptations, and are frequently studied in hematopoietic, renal, and cancer model systems. EPO is also used as a readout for hypoxia pathway activity and as a marker of cell-state changes affecting endocrine cytokine secretion.

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

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

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