BTLA CRISPR Activation Plasmid (m): sc-431543-ACT

Btla encodes B and T lymphocyte attenuator (BTLA), an inhibitory immunoreceptor of the CD28 family that restrains lymphocyte activation and helps maintain peripheral tolerance. BTLA signaling is initiated by engagement with HVEM (TNFRSF14) and recruits phosphatases through ITIM/ITSM motifs to dampen antigen receptor–proximal signaling, limiting cytokine production, proliferation, and effector differentiation. In mouse immune cells, BTLA contributes to regulation of T cell exhaustion-like programs, germinal center responses, and control of inflammatory cues within lymphoid tissues. Dysregulated BTLA pathways are broadly relevant to studies of autoimmunity, chronic inflammation, infection-driven immune remodeling, and tumor immunology in the context of immune checkpoint biology.

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

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

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