AAV with CMV7 promoter driven EGFP-T2A-CreERT2

This AAV expresses EGFP-T2A-CreERT2 driven by an ubiquitous CMV7 promoter.

The CMV7 promoter is about 0.35 kb in length and is a shortened version of the commonly used CMV promoter. CMV7 has a truncated version of the CMV enhancer. When tested in several cell lines, this promoter provides an expression level that is about 3-5 fold lower than that of the original 0.6Kb CMV promoter.

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.