TLR4 CRISPR Activation Plasmid (h): sc-400068-ACT

Human TLR4 (Toll-like receptor 4) is a pattern recognition receptor that detects lipopolysaccharide and other pathogen- and damage-associated molecular patterns at the plasma membrane. Upon ligand engagement with co-receptors such as MD-2 and CD14, TLR4 initiates MyD88- and TRIF-dependent signaling cascades that activate NF-κB, AP-1, and IRF3, driving transcriptional programs for inflammatory cytokines and type I interferon responses. These pathways coordinate innate immune activation, leukocyte recruitment, and crosstalk with adaptive immunity through antigen-presenting cells. Dysregulated TLR4 signaling has been implicated in chronic inflammatory states and immune-mediated pathology, and is widely studied in contexts including sepsis biology, atherosclerosis, metabolic inflammation, and tumor-associated inflammation.

TLR4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TLR4 expression without altering the underlying DNA sequence.

TLR4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TLR4 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 TLR4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TLR4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TLR4 locus and enabling the study of TLR4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TLR4 pathway restoration in tumor cells with silenced or reduced TLR4 expression.

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