Ribosomal Protein L5 CRISPR Activation Plasmid (h): sc-403453-ACT

RPL5 encodes ribosomal protein L5, a core component of the 60S large ribosomal subunit that binds and escorts 5S rRNA during ribosome biogenesis and supports accurate translation. Beyond its structural role in cytosolic ribosomes, RPL5 contributes to nucleolar homeostasis and links ribosomal stress to p53 pathway regulation via interactions with MDM2, influencing cell-cycle control and apoptosis. Perturbation of RPL5 can disrupt rRNA processing and global protein synthesis, affecting proliferation and differentiation programs. Altered RPL5 function or dosage has been associated with ribosomopathies and cancer-related translational dysregulation, making it relevant for studies of proteostasis and stress signaling.

Ribosomal Protein L5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RPL5 expression without altering the underlying DNA sequence.

Ribosomal Protein L5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RPL5 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 RPL5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ribosomal Protein L5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RPL5 locus and enabling the study of Ribosomal Protein L5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ribosomal Protein L5 pathway restoration in tumor cells with silenced or reduced RPL5 expression.

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