TBX5 CRISPR Activation Plasmid (m): sc-423282-ACT

Tbx5 encodes the T-box transcription factor TBX5, a sequence-specific DNA-binding regulator that orchestrates developmental gene programs controlling cardiac chamber formation, conduction system maturation, and forelimb morphogenesis in mouse. TBX5 functions in concert with cofactors such as NKX2-5 and GATA4 to regulate enhancer networks and chromatin states that shape lineage specification and tissue patterning. Its activity integrates into transcriptional control pathways governing cardiomyocyte differentiation, electrical coupling, and structural morphogenesis. Altered TBX5 dosage or regulatory function is linked to congenital heart and limb malformations, making it a key node for modeling developmental dysregulation in vivo and in stem cell–based systems.

TBX5 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Tbx5 expression without altering the underlying DNA sequence.

TBX5 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Tbx5 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 Tbx5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TBX5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Tbx5 locus and enabling the study of TBX5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TBX5 pathway restoration in tumor cells with silenced or reduced Tbx5 expression.

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