tsg 101 CRISPR Activation Plasmid (m): sc-423524-ACT

Tsg101 encodes tumor susceptibility gene 101, a core component of the ESCRT-I complex that coordinates endosomal sorting, multivesicular body biogenesis, and ubiquitin-dependent trafficking of membrane proteins. In mouse cells, tsg 101 supports lysosomal degradation pathways, receptor downregulation, and membrane remodeling events linked to cytokinesis and autophagy. Through its role in ESCRT-mediated membrane scission, Tsg101 influences exosome biogenesis and viral budding, processes frequently leveraged in models of infection and intercellular signaling. Dysregulated ESCRT function and altered Tsg101 expression have been associated with aberrant cell proliferation, genomic instability, and oncogenic phenotypes in experimental systems, making it relevant for studying cancer-related cell biology.

tsg 101 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Tsg101 expression without altering the underlying DNA sequence.

tsg 101 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Tsg101 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 Tsg101 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous tsg 101 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Tsg101 locus and enabling the study of tsg 101-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of tsg 101 pathway restoration in tumor cells with silenced or reduced Tsg101 expression.

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