AAV with GFAP(0.7) promoter driven RFP-T2A-CreERT2

This AAV expresses RFP-T2A-CreERT2 driven by an astrocyte GFAP(0.7) promoter.

This 0.7 Kb novel GFAP promoter was constructed by ligating several key regulatory elements of the 2.2Kb human GFAP promoter. It drives essentially the same expression pattern as the parental 2.2 Kb GFAP promoter but with two-fold greater activity.

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.