AAV with CamKII(0.4) promoter driven mCherry-T2A-CreERT2

This AAV expresses mCherry-T2A-CreERT2 driven by a neuron CamKII(0.4) promoter.

This 0.4 Kb promoter is derived from murine α-Calcium/calmodulin-dependent kinase II (CaMKII), a gene with expression restricted to excitatory neurons in the neocortex and hippocampus. It was shown that the promoter activity of CaMKII(0.4) is almost identical to that of CaMKII(1.3) in cortical neurons, Compared to CaMKII(1.3), this short promoter allows cloning of longer gene of interest into the AAV construct .

The short 2A peptide sequences, when cloned in-frame between two genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. This differs from conventional approaches for multiple protein expressions, such as IRES-mediated bicistronic gene expression, which has several limitations including imbalanced protein expression. The use of 2A peptide sequences alleviates these concerns, since 2A-mediated “self-cleavage” gives rise to a 1:1 ratio of the two separate proteins. Several forms of 2A peptide are commonly used: T2A (Thoseaasigna virus 2A), P2A (porcine teschovirus-1 2A), E2A (equine rhinitis A virus), and F2A (foot-and-mouth disease virus, FMDV 2A).

A mutated form of estrogen ligand-binding domain (ERT2) that binds to synthetic antagonists (such as tamoxifen or its derivative 4-hydroxy-tamoxifen) but not to circulating estrogen, was fused to the Cre recombinase (Cre) to create CreERT2 (also known as “inducible Cre”). When tamoxifen binds to CreERT2, it induces a conformational change of CreERT2, leading to its nuclear translocation, followed by Cre/Lox-mediated recombination.